Peptide variants of protein A

ABSTRACT

Z domain variants of staphylococcal protein A have significantly reduced size but possess IgG-binding affinity equivalent to the wild type Z domain. These Z domain variants are suitable for use in affinity chromatography purification of proteins and in the treatment of staphylococcic diseases.

This application is a continuation application of U.S. Ser. No.08/657,983 filed Jun. 4, 1996, now U.S. Pat. No. 6,013,763, the entiredisclosure of which is incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to the field of staphylococcal protein A, andmore particularly to the gamma-immunoglobulin binding domains of proteinA.

BACKGROUND OF THE INVENTION

Protein A from Staphylococcus aureus binds with high affinity and highspecificity to the Cγ2-Cγ3 interface region of IgG (Langone, Adv.Immunol., 32: 157-252 (1982)). Protein A also exhibits an affinity forthe Fab region of immunoglobulins that are encoded by the V_(H) genefamily, V_(H)III (Sasso et al., J. Immunol, 61: 3026-3031 (1991);Hillson et al., A. J Exp. Med., 178: 331-336 (1993)). The sequence ofthe gene coding for protein A revealed two functionally distinct regions(Uhlen et al., J. Biol. Chem., 259: 1695-1702 (1984); Lofdahl et al.,Proc. Natl. Acad Sci (USA), 80: 697-701 (1983)). The amino-terminalregion contains five highly homologous IgG-binding domains (termed E, D,A, B and C), and the carboxy terminal region anchors the protein to thecell wall and membrane. All five IgG-binding domains of protein A bindto IgG via the Fc region.

The structure of the B domain has been studied using ¹H-NMR (Torigoe etal., Biochem, 29: 8787-8793 (1990); Gouda et al., Biochem., 31:9665-9672 (1992)) and found to consist of three α-helical regions (α₁,α₂, α₃ corresponding to helices I, II and III in the NMR structure)which also are retained when bound to the Fc region of IgG (Gouda,supra). The tri-helical nature of the bound state is in contrast to theX ray crystal structure reported by Deisenhofer, Biochem., 20: 2361-2370(1981) which showed that α₁ and α₂ helices of the B domain were presentwhile the α₃ helix did not form. The X ray crystallographic analysisreported by Deisenhofer, supra, and the mutagenesis studies reported byPopplewell et al., Protein Eng., 4: 963-970 (1991) and Cedergren et al.,Protein Eng., 6: 441-448 (1993) have also identified ten residues withinα₁ and α₂ and nine residues within the Fc region of IgG whichparticipate in the protein-protein interaction.

The interaction between protein A and IgG forms the basis of manyimmunoaffinity-based purification procedures for antibodies (Hjelm, FEBSLett., 28: 73-76 (1972)), antibody fragments and antigens (Sisson andCarter, Immunol. Meth., 127: 215-22- (1990)). Bottomley et al., J.Immunol. Meth., 182: 185-192 (1995) reported that truncating B domainvariant peptides lacking the 13 C-terminal residues of the α₃ helixcauses a decrease in IgG-binding activity. Bottomley et al. alsocharacterized various mutations of residues within the α₁ and α₂ regionsfound to reduce or not affect IgG-binding activity.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a compound represented by Formula(I):

X₁-AA₆-AA₇-AA₈-AA₉-Gln-Gln-AA₁₂-AA₁₃-Phe-Tyr-Glu-Ala-Leu-His-Asp-Pro-Asn-Leu-Asn-Glu-Glu-Gln-Arg-Asn-Ala-Lys-Ile-AA₃₃-Ser-Ile-AA₃₆-Asp-Asp-X₂(SEQ ID NO:1)  (I)

where

X₁ is selected from the group consisting of H, C₁-C₆alkanoyl, andZ-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2);

where

Z is selected from the group consisting of H and C₁-C₆ alkanoyl;

AA₃ is selected from the group consisting of Asp, Arg, and Ala;

AA₄ is selected from the group consisting of Asn and Gin; and

AA₅ is selected from the group consisting of Lys, Gly, and Ser;

AA₆ is selected from the group consisting of Phe and Gly;

AA₇ is selected from the group consisting of Asn and Trp;

AA₈ is selected from the group consisting of Lys and Met;

AA₉ is selected from the group consisting of Glu, Gln, and Arg;

AA₁₂ is selected from the group consisting of Asn, Ala, and Arg;

AA₁₃ is selected from the group consisting of Ala and Arg;

AA₃₃ is selected from the group consisting of Gin and Lys;

AA₃₆ is selected from the group consisting of Lys and Arg; and

X₂ is selected from the group consisting of OR₁ and NR₁R₂ where R₁ andR₂ are independently selected from the group consisting of H,C₁-C₆alkyl, C₆-C₁₂aryl and C₆-C₁₂aryl-C₁-C₆alkyl.

In another aspect, the invention provides a compound represented byFormula (II):

where

X₁ is selected from the group consisting of H, C₁-C₆alkanoyl, andZ-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2);

where

Z is selected from the group consisting of H and C₁-C₆alkanoyl;

AA₃ is selected from the group consisting of Asp, Arg, and Ala;

AA₄ is selected from the group consisting of Asn and Gln; and

AA₅ is selected from the group consisting of Lys, Gly, and Ser;

AA₆ is selected from the group consisting of Phe and Gly;

AA₇ is selected from the group consisting of Asn and Trp;

AA₈ is selected from the group consisting of Lys and Met;

AA₉ is selected from the group consisting of Glu, Gln, and Arg;

AA₁₂ is selected from the group consisting of Asn, Ala, and Arg;

AA₁₃ is selected from the group consisting of Ala and Arg;

AA₃₃ is selected from the group consisting of Gln and Lys;

AA₃₆ is selected from the group consisting of Lys and Arg; and

X₂ is selected from the group consisting of OR₁ and NR₁R₂ where R₁ andR₂ are independently selected from the group consisting of H,C₁-C₆alkyl, C₆-C₁₂aryl and C₆-C₁₂aryl-C₁-C₆alkyl.

BRIEF DESCRIPTION OF THE FIGURES

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawings will be provided by the Patentand Trademark Office upon request and payment of the necessary fee.

FIG. 1 is a ribbon diagram of the B-domain of Protein A (purple) incomplex with the CH2, CH3 fragment of an IgG₁ (blue) taken from x-raycoordinates. Helix-3 appears disordered in the crystal structurealthough NMR experiments indicate that it is in fact helical. In thisfigure helix-3 has been modeled as a helix.

FIGS. 2A-D are ribbon diagrams showing helices-1 and helix-2 of thetruncated Z-domain modeled from the x-ray structure. FIG. 2A depictsresidues that were randomly mutated and selected from the ExofaceLibrary. Residues that were conserved as the wild-type after selectionare shown in blue and those that demonstrated strong consensus tonon-wild-type amino acids are shown in yellow. The transparent helixrepresents the position occupied by helix-3 in the native Z domain; itwas modeled as shown in FIG. 1. Replacement residues were aligned on theC_(a), C_(b) vector of the wild type residue. FIG. 2B depicts residues(colored as in FIG. 2A) that were randomly mutated and selected from theIntraface Library. FIG. 2C depicts residues (colored as in FIG. 2A) thatwere randomly mutated and selected from the five Interface Libraries.Interface library 3A covers residues which are not depicted in thisstructure. FIG. 2D depicts an overhead view of the Interface Librarieslooking down the helical axis.

FIGS. 3A-B are graphs representing the CD spectra of the starting38-residue peptide, and the intraface and interface optimized peptides(FIG. 3A) and the extent of helicity calculated for these peptidesdirectly from the CD spectra (FIG. 3B). Data was collected and curvesfit as described in the Materials and Methods section in Example 1below. These values can be compared to the theoretical maximum value asdetermined by inspection of the structure of the first two-helices inthe intact B-domain.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS A. Definitions

Terms used in the claims and specification are defined as set forthbelow unless otherwise specified.

The terms “Z domain”, “Z domain of protein A”, “wild type Z domain”,etc., are used interchangeably herein and mean the three helix, 59 aminoacid peptide reported by Nilsson et al., Protein Engng., 1: 107-113(1987) that spans a portion of the IgG-binding B domain ofstaphylococcal protein A, and has the amino acid sequence:

AVDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK (SEQ IDNO:4)

The term “C₁-C₆ alkyl” means a branched, unbranched or cyclic, saturatedaliphatic hydrocarbon substituent, having the number of carbon atomsspecified. Representative examples of these alkyl radicals includemethyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl,n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl,2,2-dimethylbutyl, cyclohexyl and the like. The terms “lower alkyl” and“C₁-C₆alkyl” are synonymous and used interchangeably. A preferred “C₁-C₆alkyl” group is methyl.

The term “C₁-C₆ alkanoyl” encompasses groups such as formyl, acetyl,propionyl, butyryl, isobutyryl, valeryl, isovaleryl, pivaloyl, caproyl,and the like.

The term “C₆-C₁₂aryl” means a homocyclic hydrocarbon aromatic radical,whether or not fused, having the number of carbon atoms designated.Preferred aryl groups include phenyl, napthyl, biphenyl, phenanthrenyl,naphthacenyl, and the like (see e.g. Lang's Handbook of Chemistry (Dean,J. A., ed) 13th ed. Table 7-2 [1985]).

The term “C₆-C₁₂aryl-C₁-C₆alkyl” means one, two, or three aryl groupshaving the number of carbon atoms designated, appended to an alkylradical having the number of carbon atoms designated including but notlimited to benzyl, naphthylmethyl, phenethyl, benzyhydryl(diphenylmethyl), fluorenyl, trityl, and the like. A preferred arylalkylgroup is the benzyl group.

Amino acids and amino acid residues described herein may be referred toaccording to the accepted one or three letter code provided in the tablebelow. Unless otherwise specified, these amino acids or residues are ofthe naturally occurring L stereoisomer form.

Three-Letter Common Name One-Letter Symbol Symbol Alanine A Ala ArginineR Arg Asparagine N Asn Aspartic acid D Asp Cysteine C Cys Glutamine QGln Glutamic acid E Glu Glycine G Gly Histidine H His Isoleucine I IleLeucine L Leu Lysine K Lys Methionine M Met Phenylalanine F Phe ProlineP Pro Serine S Ser Threonine T Thr Tryptophan W Trp Tyrosine Y Tyr

In general, unless otherwise specified, the abbreviations used for thedesignation of amino acids and the protective groups used therefor arebased on the recommendations of the IUPAC-IUB Commission of BiochemicalNomenclature (Biochemistry, 11: 1726-1732 (1972)).

As used herein, the terms “desired protein”, “desired polypeptide”,“selected polypeptide” or “selected protein” are used interchangeablyand refer generally to any peptide or protein having more than about 5amino acids. The polypeptides may be homologous to, or preferably, maybe exogenous, meaning that they are heterologous, i.e., foreign, to therecombinant host cell in which the polypeptide is expressed, such as ahuman protein or a yeast protein produced in bacterial host cells.Preferably, mammalian polypeptides (polypeptides that were originallyderived from a mammalian organism) are used.

Examples of mammalian polypeptides include, but are not limited to,molecules such as, e.g., renin, a growth hormone, including human growthhormone; bovine growth hormone; growth hormone releasing factor;parathyroid hormone; thyroid stimulating hormone; lipoproteins;alpha-1-antitrypsin; insulin A-chain; insulin B-chain; proinsulin;follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon;clotting factors such as factor VIIIC, factor IX, tissue factor, and vonWillebrands factor; anti-clotting factors such as Protein C; atrialnatriuretic factor; lung surfactant; a plasminogen activator, such asurokinase or human urine or tissue-type plasminogen activator (t-PA);bombesin; thrombin; hemopoietic growth factor; tumor necrosisfactor-alpha and -beta; enkephalinase; RANTES (regulated on activationnormally T-cell expressed and secreted); human macrophage inflammatoryprotein (MIP-1-alpha); a serum albumin such as human serum albumin;mullerian-inhibiting substance; relaxin A-chain; relaxin B-chain;prorelaxin; mouse gonadotropin-associated peptide; a microbial protein,such as beta-lactamase; DNase; inhibin; activin; vascular endothelialgrowth factor (VEGF); receptors for hormones or growth factors;integrin; protein A or D; rheumatoid factors; a neurotrophic factor suchas bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or-6 (NT-3, NT-4, NT-5, or NT-6), or a nerve growth factor such as NGF-β;platelet-derived growth factor (PDGF); fibroblast growth factor such asaFGF and bFGF; epidermal growth factor (EGF); transforming growth factor(TGF) such as TGF-alpha and TGF-beta, including TGF-β1, TGF-β2, TGF-β3,TGF-β4, or TGF-β5; insulin-like growth factor-I and -II (IGF-I andIGF-II); des(1-3)-IGF-I (brain IGF-I), insulin-like growth factorbinding proteins; CD proteins such as CD-3, CD-4, CD-8, and CD-19;erythropoietin; osteoinductive factors; immunotoxins; a bonemorphogenetic protein (BMP); an interferon such as interferon-alpha,-beta, and -gamma; colony stimulating factors (CSFs), e.g., M-CSF,GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 to IL-10; superoxidedismutase; T-cell receptors; surface membrane proteins; decayaccelerating factor; viral antigen such as, for example, a portion ofthe AIDS envelope; transport proteins; homing receptors; addressins;regulatory proteins; antibodies; and fragments of any of theabove-listed polypeptides.

B. General Methods

In general, the invention provides variants of the Z domain ofstaphylococcal protein A for use in affinity purification of proteins,such as gamma-immunoglobulins (IgGs) and IgG-carrying fusion proteins(immunoadhesins), for use in cell separation techniques for IgG-bearingcells, and for use in the treatment of staphylococcal infections. The Zdomain variants of the invention provide the practitioner with moleculesthat are approximately 50% smaller than the wild type Z domain whileretaining IgG-binding activities that are equivalent or comparable tothat of the intact, wild type Z domain of protein A. In the case ofprotein purification, the Z domain variants can be fused to recombinantproteins as affinity purification handles capable of achievingsingle-step, quantitative purification of fusion proteins by IgG-ligandchromatography. The reduced size of the present Z domain variantsconfers the advantages of lower cost (e.g. conservation of biosyntheticor synthetic chemical production resources), greater versatility andreduced influence on the higher order structure of the parent proteinand its activity in fusion protein form. Likewise, the smaller size ofthe present Z domain variants facilitates their use as ligands inaffinity chromatography of gamma-immunoglobulins (IgGs), IgG fusionproteins and IgG-bearing cells with advantages such as greater stability(enabling prolonged use as ligands in affinity chromatography), smallersize (providing higher density ligand on solid supports for higherbinding capacity/yield of affinity chromatography), and greater costefficiency associated therewith, and reduces the liklihood ofcopurification of contaminants which interact with areas of the Z domainthat do not directly contact the protein A-binding determinants of IgG.Additionally, the peptides of the invention are less expensive toproduce as pharmaceutical agents and are less likely to elicit anundesired (e.g. inactivating) immune response than are full length, wildtype Z domain peptides.

I. Preferred Embodiments

In one aspect, the invention provides for Z domain variant peptideswhich lack the α₃ helix present in the full length, wild type Z domain,causing a loss in the conformational stability and IgG-binding activityof the variant peptides, and which are further engineered to largelyrestore the lost stability and IgG-binding activity by improving thequality of intramolecular contacts in the remaining peptide scaffoldthrough select amino acid substitutions. Accordingly, the inventionprovides linear Z domain variant peptides represented by Formula (I):

X₁-AA₆-AA₇-AA₈AA-₉-Gln-Gln-AA₁₂-AA₁₃-Phe-Tyr-Glu-Ala-Leu-His-Asp-Pro-Asn-Leu-Asn-Glu-Glu-Gln-Arg-Asn-Ala-Lys-Ile-AA₃₃-Ser-Ile-AA₃₆-Asp-Asp-X₂(SEQ ID NO:1)  (I)

where

X₁ is selected from the group consisting of H, C₁-C₆alkanoyl, andZ-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2);

where

Z is selected from the group consisting of H and C₁-C₆ alkanoyl;

AA₃ is selected from the group consisting of Asp, Arg, and Ala;

AA₄ is selected from the group consisting of Asn and Gln; and

AA₅ is selected from the group consisting of Lys, Gly, and Ser;

AA₆ is selected from the group consisting of Phe and Gly;

AA₇ is selected from the group consisting of Asn and Trp;

AA₈ is selected from the group consisting of Lys and Met;

AA₉ is selected from the group consisting of Glu, Gln, and Arg;

AA₁₂ is selected from the group consisting of Asn, Ala, and Arg;

AA₁₃ is selected from the group consisting of Ala and Arg;

AA₃₃ is selected from the group consisting of Gln and Lys;

AA₃₆ is selected from the group consisting of Lys and Arg; and

X₂ is selected from the group consisting of OR₁ and NR₁R₂ where R₁ andR₂ are independently selected from the group consisting of H,C₁-C₆alkyl, C₆-C₁₂aryl and C₆-C₁₂aryl-C₁-C₆alkyl.

In one embodiment, the invention provides peptides of Formula (I)wherein X₁ is Z-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ is Arg, and AA₅is Gly.

In another embodiment, the invention provides peptides of Formula (I)wherein X₁ is Z-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ is Arg, AA₅ isGly, AA₄ is Asn, AA₆ is Phe, AA₇ is Asn, AA₈ is Lys, AA₉ is Glu, AA₁₂ isAsn, AA₁₃ is Arg, AA₃₃ is Gln, and AA₃₆ is Lys.

In yet another embodiment, the invention provides peptides of Formula(I) wherein X₁ is Z-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ is Arg, AA₅is Gly, and AA₁₂ is Ala.

Further provided herein are peptides of Formula (I) wherein X₁ isZ-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ is Arg, AA₅ is Gly, AA₄ is Asn,AA₆ is Phe, AA₇ is Asn, AA₈ is Lys, AA₉ is Glu, AA₁₂ is Ala, AA₁₃ isArg, AA₃₃ is Gln, and AA₃₆ is Lys.

In still another embodiment, the invention provides peptides of Formula(I) wherein AA₈ is Met and AA₉ is Gln.

Also provided herein are peptides of Formula (I) wherein AA₈ is Met, AA₉is Gln, X₁ is Z-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ is Asp, AA₄ isAsn, AA₅ is Lys, AA₆ is Phe, AA₇ is Asn, AA₁₂ is Asn, AA₁₃ is Arg, AA₃₃is Gln, and AA₃₆ is Lys.

Additionally provided herein are peptides of Formula (I) wherein AA₈ isMet, AA₉ is Gln, and AA₁₂ is Ala.

In a further aspect, the invention provides peptides of Formula (I)wherein AA₈ is Met, AA₉ is Gln, X₁ is Z-Ala-Val-AA₃-AA₄-AA₅ (SEQ IDNO:2), AA₃ is Asp, AA₄ is Asn, AA₅ is Lys, AA₆ is Phe, AA₇ is Asn, AA₁₂is Ala, AA₁₃ is Arg, AA₃₃ is Gln, and AA₃₆ is Lys.

In an additional embodiment, the invention provides peptides of Formula(I) wherein AA₁₂ is Arg and AA₁₃ is Ala.

In a further embodiment, the invention provides peptides of Formula (I)wherein AA₁₂ is Arg, AA₁₃ is Ala, X₁ is Z-Ala-Val-AA₃-AA₄-AA₅ (SEQ IDNO:2), AA₃ is Asp, AA₄ is Asn, AA₅ is Lys, AA₆ is Phe, AA₇ is Asn, AA₈is Lys, AA₉ is Glu, AA₃₃ is Gln, and AA₃₆ is Lys.

Also encompassed within the scope of the invention are peptides ofFormula (I) wherein AA₁₂ is Ala and AA₁₃ is Ala.

Further encompassed within the scope of the invention are peptides ofFormula (I) wherein AA₁₂ is Ala, AA₁₃ is Ala, X₁ isZ-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ is Asp, AA₄ is Asn, AA₅ is Lys,AA₆ is Phe, AA₇ is Asn, AA₈ is Lys, AA₉ is Glu, AA₃₃ is Gln; and AA₃₆ isLys.

In another aspect, the invention provides a compound selected from thegroup consisting ofZ-Ala-Val-Asp-Asn-Lys-Phe-Asn-Lys-Glu-Gln-Gln-Asn-Arg-Phe-Tyr-Glu-Ala-Leu-His-Asp-Pro-Asn-Leu-Asn-Glu-Glu-Gln-Arg-Asn-Ala-Lys-Ile-Gln-Ser-Ile-Lys-Asp-Asp-X₂(SEQ ID NO:5);

Z-Ala-Val-Arg-Asn-Gly-Phe-Asn-Lys-Glu-Gln-Gln-Asn-Arg-Phe-Tyr-Glu-Ala-Leu-His-Asp-Pro-Asn-Leu-Asn-Glu-Glu-Gln-Arg-Asn-Ala-Lys-Ile-Gln-Ser-Ile-Lys-Asp-Asp-X₂(SEQ ID NO:6);

Z-Ala-Val-Ala-Gln-Ser-Phe-Asn-Lys-Glu-Gln-Gln-Asn-Arg-Phe-Tyr-Glu-Ala-Leu-His-Asp-Pro-Asn-Leu-Asn-Glu-Glu-Gln-Arg-Asn-Ala-Lys-Ile-Gln-Ser-Ile-Lys-Asp-Asp-X₂(SEQ ID NO:7);

Z-Ala-Val-Asp-Asn-Lys-Phe-Asn-Met-Gln-Gln-Gln-Asn-Arg-Phe-Tyr-Glu-Ala-Leu-His-Asp-Pro-Asn-Leu-Asn-Glu-Glu-Gln-Arg-Asn-Ala-Lys-Ile-Gln-Ser-Ile-Lys-Asp-Asp-X₂(SEQ ID NO:8);

Z-Ala-Val-Asp-Asn-Lys-Gly-Trp-Met-Arg-Gln-Gln-Asn-Arg-Phe-Tyr-Glu-Ala-Leu-His-Asp-Pro-Asn-Leu-Asn-Glu-Glu-Gln-Arg-Asn-Ala-Lys-Ile-Gln-Ser-Ile-Lys-Asp-Asp-X₂(SEQ ID NO:9);

Z-Ala-Val-Asp-Asn-Lys-Phe-Asn-Lys-Glu-Gln-Gln-Arg-Arg-Phe-Tyr-Glu-Ala-Leu-His-Asp-Pro-Asn-Leu-Asn-Glu-Glu-Gln-Arg-Asn-Ala-Lys-Ile-Gln-Ser-Ile-Lys-Asp-Asp-X₂(SEQ ID NO:10);

Z-Ala-Val-Asp-Asn-Lys-Phe-Asn-Lys-Glu-Gln-Gln-Arg-Ala-Phe-Tyr-Glu-Ala-Leu-His-Asp-Pro-Asn-Leu-Asn-Glu-Glu-Gln-Arg-Asn-Ala-Lys-Ile-Gln-Ser-Ile-Lys-Asp-Asp-X₂(SEQ ID NO:11);

Z-Ala-Val-Asp-Asn-Lys-Phe-Asn-Lys-Glu-Gln-Gln-Asn-Arg-Phe-Tyr-Glu-Ala-Leu-His-Asp-Pro-Asn-Leu-Asn-Glu-Glu-Gln-Arg-Asn-Ala-Lys-Ile-Lys-Ser-Ile-Arg-Asp-Asp-X₂(SEQ ID NO:12);

Z-Ala-Val-Arg-Asn-Gly-Phe-Asn-Met-Gln-Gln-Gln-Arg-Arg-Phe-Tyr-Glu-Ala-Leu-His-Asp-Pro-Asn-Leu-Asn-Glu-Glu-Gln-Arg-Asn-Ala-Lys-Ile-Lys-Ser-Ile-Arg-Asp-Asp-X₂(SEQ ID NO:13);

Z-Ala-Val-Ala-Gln-Ser-Phe-Asn-Met-Gln-Gln-Gln-Arg-Arg-Phe-Tyr-Glu-Ala-Leu-His-Asp-Pro-Asn-Leu-Asn-Glu-Glu-Gln-Arg-Asn-Ala-Lys-Ile-Lys-Ser-Ile-Arg-Asp-Asp-X₂(SEQ ID NO:14); and

X₁-Phe-Asn-Met-Gln-Gln-Gln-Arg-Arg-Phe-Tyr-Glu-Ala-Leu-His-Asp-Pro-Asn-Leu-Asn-Glu-Glu-Gln-Arg-Asn-Ala-Lys-Ile-Lys-Ser-Ile-Arg-Asp-Asp-X₂(SEQ ID NO: 15);

where

Z is selected from the group consisting of H and C₁-C₆alkanoyl;

X₁ is selected from the group consisting of H and C₁-C₆ alkanoyl; and

X₂ is selected from the group consisting of OR₁ and NR₁R₂ where R₁ andR₂ are independently selected from the group consisting of H, C₁-C₆alkyl, C₆-C₁₂aryl and C₆-C₁₂aryl-C₁-C₆alkyl.

In an additional aspect, the invention provides a compound selected fromthe group consisting of

Z-Ala-Val-Asp-Asn-Lys-Phe-Asn-Lys-Glu-Gln-Gln-Ala-Arg-Phe-Tyr-Glu-Ala-Leu-His-Asp-Pro-Asn-Leu-Asn-Glu-Glu-Gln-Arg-Asn-Ala-Lys-Ile-Gln-Ser-Ile-Lys-Asp-Asp-X₂(SEQ ID NO:16);

Z-Ala-Val-Arg-Asn-Gly-Phe-Asn-Lys-Glu-Gln-Gln-Ala-Arg-Phe-Tyr-Glu-Ala-Leu-His-Asp-Pro-Asn-Leu-Asn-Glu-Glu-Gln-Arg-Asn-Ala-Lys-Ile-Gln-Ser-Ile-Lys-Asp-Asp-X₂(SEQ ID NO:17);

Z-Ala-Val-Ala-Gln-Ser-Phe-Asn-Lys-Glu-Gln-Gln-Ala-Arg-Phe-Tyr-Glu-Ala-Leu-His-Asp-Pro-Asn-Leu-Asn-Glu-Glu-Gln-Arg-Asn-Ala-Lys-Ile-Gln-Ser-Ile-Lys-Asp-Asp-X₂(SEQ ID NO:18);

Z-Ala-Val-Asp-Asn-Lys-Phe-Asn-Met-Gln-Gln-Gln-Ala-Arg-Phe-Tyr-Glu-Ala-Leu-His-Asp-Pro-Asn-Leu-Asn-Glu-Glu-Gln-Arg-Asn-Ala-Lys-Ile-Gln-Ser-Ile-Lys-Asp-Asp-X₂(SEQ ID NO:19);

Z-Ala-Val-Asp-Asn-Lys-Gly-Trp-Met-Arg-Gln-Gln-Ala-Arg-Phe-Tyr-Glu-Ala-Leu-His-Asp-Pro-Asn-Leu-Asn-Glu-Glu-Gln-Arg-Asn-Ala-Lys-Ile-Gln-Ser-Ile-Lys-Asp-Asp-X₂(SEQ ID NO:20);

Z-Ala-Val-Asp-Asn-Lys-Phe-Asn-Lys-Glu-Gln-Gln-Ala-Ala-Phe-Tyr-Glu-Ala-Leu-His-Asp-Pro-Asn-Leu-Asn-Glu-Glu-Gln-Arg-Asn-Ala-Lys-Ile-Gln-Ser-Ile-Lys-Asp-Asp-X₂(SEQ ID NO:21);

Z-Ala-Val-Asp-Asn-Lys-Phe-Asn-Lys-Glu-Gln-Gln-Ala-Arg-Phe-Tyr-Glu-Ala-Leu-His-Asp-Pro-Asn-Leu-Asn-Glu-Glu-Gln-Arg-Asn-Ala-Lys-Ile-Lys-Ser-Ile-Arg-Asp-Asp-X₂(SEQ ID NO:22);

Z-Ala-Val-Arg-Asn-Gly-Phe-Asn-Met-Gln-Gln-Gln-Ala-Arg-Phe-Tyr-Glu-Ala-Leu-His-Asp-Pro-Asn-Leu-Asn-Glu-Glu-Gln-Arg-Asn-Ala-Lys-Ile-Lys-Ser-Ile-Arg-Asp-Asp-X₂(SEQ ID NO:23);

Z-Ala-Val-Ala-Gln-Ser-Phe-Asn-Met-Gln-Gln-Gln-Ala-Arg-Phe-Tyr-Glu-Ala-Leu-His-Asp-Pro-Asn-Leu-Asn-Glu-Glu-Gln-Arg-Asn-Ala-Lys-Ile-Lys-Ser-Ile-Arg-Asp-Asp-X₂(SEQ ID NO:24); and

X₁-Phe-Asn-Met-Gln-Gln-Gln-Ala-Arg-Phe-Tyr-Glu-Ala-Leu-His-Asp-Pro-Asn-Leu-Asn-Glu-Glu-Gln-Arg-Asn-Ala-Lys-Ile-Lys-Ser-Ile-Arg-Asp-Asp-X₂(SEQ ID NO:25);

where

Z is selected from the group consisting of H and C₁-C₆ alkanoyl;

X₁ is selected from the group consisting of H and C₁-C₆alkanoyl; and

X₂ is selected from the group consisting of OR₁ and NR₁R₂ where R₁ andR₂ are independently selected from the group consisting of H, C₁-C₆alkyl, C₆-C₁₂aryl and C₆-C₁₂aryl-C₁-C₆alkyl.

In a preferred embodiment, the invention provides peptides of Formula(I) wherein X₁ is Z-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ is Ala, AA₄is Gln, and AA₅ is Ser.

In another preferred embodiment, the invention provides peptides ofFormula (I) wherein X₁ is Z-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ isAla, AA₄ is Gln, AA₅ is Ser, AA₆ is Phe, AA₇ is Asn, AA₈ is Lys, AA₉ isGlu, AA₁₂ is Asn, AA₁₃ is Arg, AA₃₃ is Gln, and AA₃₆ is Lys.

In yet another aspect, the invention provides peptides of Formula (I)wherein X₁ is Z-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ is Ala, AA₄ isGln, AA₅ is Ser, and AA₁₂ is Ala.

In still another aspect, the invention provides peptides of Formula (I)wherein X₁ is Z-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ is Ala, AA₄isGln, AA₅ is Ser, AA₆ is Phe, AA₇ is Asn, AA₈ is Lys, AA₉ is Glu, AA₁₂ isAla, AA₁₃ is Arg, AA₃₃ is Gln, and AA₃₆ is Lys.

In yet another preferred embodiment, the invention provides peptides ofFormula (I) wherein X₁ is AA₆ is Gly, AA₇ is Trp, AA₈ is Met, and AA₉ isArg.

In still another preferred embodiment, the invention provides peptidesof Formula (I) wherein X₁ is Z-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₆ isGly, AA₇ is Trp, AA₈ is Met, AA₉ is Arg, AA₃ is Asp, AA₄ is Asn, AA₅ isLys, AA₁₂ is Asn, AA₁₃ is Arg, AA₃₃ is Gln; and AA₃₆ is Lys.

Also encompassed herein are peptides of Formula (I) wherein AA₆ is Gly,AA₇ is Trp, AA₈ is Met, AA₉ is Arg, and AA₁₂ is Ala.

Further encompassed herein are peptides of Formula (I) wherein X₁ isZ-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₆ is Gly, AA₇ is Trp, AA₈ is Met,AA₉ is Arg, AA₃ is Asp, AA₄ is Asn, AA₅ is Lys, AA₁₂ is Ala, AA₁₃ isArg, AA₃₃ is Gln; and AA₃₆ is Lys.

In a further preferred embodiment, the invention provides peptides ofFormula (I) wherein AA₁₂ is Arg.

Also preferred are peptides of Formula (I) wherein X₁ isZ-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₁₂ is Arg, AA₃ is Asp, AA₄ isAsn, AA₅ is Lys, AA₆ is Phe, AA₇ is Asn, AA₈ is Lys, AA₉ is Glu, AA₁₃ isArg, AA₃₃ is Gln, and AA₃₆ is Lys.

Further encompassed within the scope of the invention are peptides ofFormula (I) wherein AA₁₂ is Ala.

Additionally encompassed within the scope of the invention are peptidesof Formula (I) wherein X₁ is Z-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₁₂is Ala, AA₃ is Asp, AA₄ is Asn, AA₅ is Lys, AA₆ is Phe, AA₇ is Asn, AA₈is Lys, AA₉ is Glu, AA₁₃ is Arg, AA₃₃ is Gln, and AA₃₆ is Lys.

In an additional preferred embodiment, the invention provides peptidesof Formula (I) wherein AA₃₃ is Lys and AA₃₆ is Arg.

In still another preferred embodiment, the invention provides peptidesof Formula (I) wherein X₁ is Z-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃₃is Lys, AA₃₆ is Arg, AA₃ is Asp, AA₄ is Asn, AA₅ is Lys, AA₆ is Phe, AA₇is Asn, AA₈ is Lys, AA₉ is Glu, AA₁₂ is Asn, and AA₁₃ is Arg.

Further encompassed herein are peptides of Formula (I) wherein AA₃₃ isLys, AA₃₆ is Arg, and AA₁₂ is Ala.

Additionally encompassed herein are peptides of Formula (I) wherein X₁is Z-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃₃ is Lys, AA₃₆ is Arg, AA₃ isAsp, AA₄ is Asn, AA₅ is Lys, AA₆ is Phe, AA₇ is Asn, AA₈ is Lys, AA₉ isGlu, AA₁₂ is Ala, and AA₁₃ is Arg.

In yet another preferred embodiment, the invention provides peptides ofFormula (I) wherein X₁ is Z-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ isArg, AA₅ is Gly, AA₈ is Met, AA₉ is Gln, AA₁₂ is Arg, AA₃₃ is Lys, andAA₃₆ is Arg.

In a more preferred embodiment, the invention provides peptides ofFormula (I) wherein X₁ is Z-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ isArg, AA₅ is Gly, AA, is Met, AA₉ is Gln, AA₁₂ is Arg, AA₃₃ is Lys, AA₃₆is Arg, AA₄ is Asn, AA₆ is Phe, AA₇is Asn, and AA₃ is Arg.

Also provided herein are peptides of Formula (I) wherein X₁ isZ-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ is Arg, AA₅ is Gly, AA₈ is Met,AA₉ is Gln, AA₁₂ is Ala, AA₃₃ is Lys, and AA₃₆ is Arg.

Further provided herein are peptides of Formula (I) wherein X₁ isZ-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ is Arg, AA₅ is Gly, AA₈ is Met,AA₉ is Gln, AA₁₂ is Ala, AA₃₃ is Lys, AA₃₆ is Arg, AA₄ is Asn, AA₆ isPhe, AA₇ is Asn, and AA₁₃ is Arg.

In an additional preferred embodiment, the invention provides peptidesof Formula (I) wherein X₁ is Z-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ isAla, AA₄ is Gln, AA₅ is Ser, AA₈ is Met, AA₉ is Gln, AA₁₂ is Arg, AA₃₃is Lys, and AA₃₆ is Arg.

In a more preferred embodiment, the invention provides peptides ofFormula (I) wherein X₁ is Z-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ isAla, AA₄ is Gln, AA₅ is Ser, AA₈ is Met, AA₉ is Gln, AA₁₂ is Arg, AA₃₃is Lys, AA₃₆ is Arg, AA₆ is Phe, AA₇ is Asn, and AA₁₃ is Arg.

Additionally provided herein are peptides of Formula (I) wherein X₁ isZ-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ is Ala, AA₄ is Gln, AA₅ is Ser,AA₈ is Met, AA₉ is Gln, AA₁₂ is Ala, AA₃₃ is Lys, and AA₃₆ is Arg.

In another embodiment, the invention provides peptides of Formula (I)wherein X, is Z-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ is Ala, AA₄ isGln, AA₅ is Ser, AA₈ is Met, AA₉ is Gln, AA₁₂ is Ala, AA₃₃ is Lys, AA₃₆is Arg, AA₆ is Phe, AA₇ is Asn, and AA₁₃ is Arg.

In a particularly preferred embodiment, the invention provides peptidesof Formula (I) wherein X₁ is H or C₁-C₆alkanoyl, AA₈ is Met, AA₉ is Gln,AA₁₂ is Arg, AA₃₃ is Lys, and AA₃₆ is Arg.

In an even more preferred embodiment, the invention provides peptides ofFormula (I) wherein X₁ is H or C₁-C₆alkanoyl, AA₈ is Met, AA₉ is Gln,AA₁₂ is Arg, AA₃₃ is Lys, AA₃₆ is Arg, AA₆ is Phe, AA₇ is Asn, and AA₃is Arg.

In yet another embodiment, the invention provides peptides of Formula(I) wherein X₁ is H or C₁-C₆alkanoyl, AA₈ is Met, AA₉ is Gln, AA₁₂ isAla, AA₃₃ is Lys, and AA₃₆ is Arg.

In still another embodiment, the invention provides peptides of Formula(I) wherein X₁ is H or C₁-C₆alkanoyl, AA₈ is Met, AA₉ is Gln, AA₁₂ isAla, AA₃₃ is Lys, AA₃₆ is Arg, AA₆ is Phe, AA₇ is Asn, and AA₁₃ is Arg.

In another aspect, the invention provides peptides represented byFormula (Ia):

X₁-AA₆-AA₇-AA₈-AA₉-Gln-Gln-AA₁₂-AA₁₃-Phe-Tyr-Glu-Ala-Leu-His-Asp-Pro-Asn-Leu-Asn-Glu-Glu-Gln-Arg-Asn-Ala-Lys-Ile-AA₃₃-Ser-Ile-AA₃₆-Asp-Asp(SEQ ID NO:1),  (Ia)

where

X₁ is absent or is Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2);

where

AA₃ is selected from the group consisting of Asp, Arg, and Ala;

AA₄ is selected from the group consisting of Asn and Gln; and

AA₅ is selected from the group consisting of Lys, Gly, and Ser;

AA₆ is selected from the group consisting of Phe and Gly;

AA₇ is selected from the group consisting of Asn and Trp;

AA₈ is selected from the group consisting of Lys and Met;

AA₉ is selected from the group consisting of Glu, Gln, and Arg;

AA₁₂ is selected from the group consisting of Asn, Ala, and Arg;

AA₁₃ is selected from the group consisting of Ala and Arg;

AA₃₃ is selected from the group consisting of Gln and Lys; and

AA₃₆ is selected from the group consisting of Lys and Arg.

In one embodiment, the invention provides peptides of Formula (Ia)wherein X₁ is Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ is Arg, and AA₅ isGly.

In another embodiment, the invention provides peptides of Formula (Ia)wherein X₁ is Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ is Arg, AA₅ is Gly,AA₄ is Asn, AA₆ is Phe, AA₇ is Asn, AA₈ is Lys, AA₉ is Glu, AA₁₂ is Asn,AA₁₃ is Arg, AA₃₃ is Gln, and AA₃₆ is Lys.

In yet another embodiment, the invention provides peptides of Formula(Ia) wherein X₁ is Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ is Arg, AA₅ isGly, and AA₁₂ is Ala.

Further provided herein are peptides of Formula (Ia) wherein X₁ isAla-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ is Arg, AA₅ is Gly, AA₄ is Asn,AA₆ is Phe, AA₇ is Asn, AA₈ is Lys, AA₉ is Glu, AA₁₂ is Ala, AA₁₃ isArg, AA₃₃ is Gln, and AA₃₆ is Lys.

In still another embodiment, the invention provides peptides of Formula(Ia) wherein AA₈ is Met and AA₉ is Gln.

Also provided herein are peptides of Formula (Ia) wherein AA₈ is Met,AA₉ is Gln, X₁ is Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ is Asp, AA₄ isAsn, AA₅ is Lys, AA₆ is Phe, AA₇ is Asn, AA₁₂ is Asn, AA₁₃ is Arg, AA₃₃is Gln, and AA₃₆ is Lys.

Additionally provided herein are peptides of Formula (Ia) wherein AA₈ isMet, AA₉ is Gln, and AA₁₂ is Ala.

In a further aspect, the invention provides peptides of Formula (la)wherein AA₈ is Met, AA₉ is Gln, X₁ is Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2),AA₃ is Asp, AA₄ is Asn, AA₅ is Lys, AA₆ is Phe, AA₇ is Asn, AA₁₂ is Ala,AA₁₃ is Arg, AA₃₃ is Gln, and AA₃₆ is Lys.

In an additional embodiment, the invention provides peptides of Formula(Ia) wherein AA₁₂ is Arg and AA₁₃ is Ala.

In a further embodiment, the invention provides peptides of Formula (Ia)wherein AA₁₂ is Arg, AA₁₃ is Ala, X₁ is Ala-Val-AA₃-AA₄-AA₅ (SEQ IDNO:2), AA₃ is Asp, AA₄ is Asn, AA₅ is Lys, AA₆ is Phe, AA₇ is Asn,AA_(8 is) Lys, AA₉ is Glu, AA₃₃ is Gln, and AA₃₆ is Lys.

Also encompassed within the scope of the invention are peptides ofFormula (Ia) wherein AA₁₂ is Ala and AA₁₃ is Ala.

Further encompassed within the scope of the invention are peptides ofFormula (Ia) wherein AA₁₂ is Ala, AA₁₃ is Ala, X₁ is Ala-Val-AA₃-AA₄-AA₅(SEQ ID NO:2), AA₃ is Asp, AA₄ is Asn, AA₅ is Lys, AA₆ is Phe, AA₇ isAsn, AA₈ is Lys, AA₉ is Glu, AA₃₃ is Gln; and AA₃₆ is Lys.

In another aspect, the invention provides a compound selected from thegroup consisting of

Ala-Val-Asp-Asn-Lys-Phe-Asn-Lys-Glu-Gln-Gln-Asn-Arg-Phe-Tyr-Glu-Ala-Leu-His-Asp-Pro-Asn-Leu-Asn-Glu-Glu-Gln-Arg-Asn-Ala-Lys-Ile-Gln-Ser-Ile-Lys-Asp-Asp(SEQ ID NO:5);

Ala-Val-Arg-Asn-Gly-Phe-Asn-Lys-Glu-Gln-Gln-Asn-Arg-Phe-Tyr-Glu-Ala-Leu-His-Asp-Pro-Asn-Leu-Asn-Glu-Glu-Gln-Arg-Asn-Ala-Lys-Ile-Gln-Ser-Ile-Lys-Asp-Asp(SEQ ID NO:6);

Ala-Val-Ala-Gln-Ser-Phe-Asn-Lys-Glu-Gln-Gln-Asn-Arg-Phe-Tyr-Glu-Ala-Leu-His-Asp-Pro-Asn-Leu-Asn-Glu-Glu-Gln-Arg-Asn-Ala-Lys-Ile-Gln-Ser-Ile-Lys-Asp-Asp(SEQ ID NO:7);

Ala-Val-Asp-Asn-Lys-Phe-Asn-Met-Gln-Gln-Gln-Asn-Arg-Phe-Tyr-Glu-Ala-Leu-His-Asp-Pro-Asn-Leu-Asn-Glu-Glu-Gln-Arg-Asn-Ala-Lys-Ile-Gln-Ser-Ile-Lys-Asp-Asp(SEQ ID NO:8);

Ala-Val-Asp-Asn-Lys-Gly-Trp-Met-Arg-Gln-Gln-Asn-Arg-Phe-Tyr-Glu-Ala-Leu-His-Asp-Pro-Asn-Leu-Asn-Glu-Glu-Gln-Arg-Asn-Ala-Lys-Ile-Gln-Ser-Ile-Lys-Asp-Asp(SEQ ID NO:9);

Ala-Val-Asp-Asn-Lys-Phe-Asn-Lys-Glu-Gln-Gln-Arg-Arg-Phe-Tyr-Glu-Ala-Leu-His-Asp-Pro-Asn-Leu-Asn-Glu-Glu-Gln-Arg-Asn-Ala-Lys-Ile-Gln-Ser-Ile-Lys-Asp-Asp(SEQ ID NO:10);

Ala-Val-Asp-Asn-Lys-Phe-Asn-Lys-Glu-Gln-Gln-Arg-Ala-Phe-Tyr-Glu-Ala-Leu-His-Asp-Pro-Asn-Leu-Asn-Glu-Glu-Gln-Arg-Asn-Ala-Lys-Ile-Gln-Ser-Ile-Lys-Asp-Asp(SEQ ID NO:11);

Ala-Val-Asp-Asn-Lys-Phe-Asn-Lys-Glu-Gln-Gln-Asn-Arg-Phe-Tyr-Glu-Ala-Leu-His-Asp-Pro-Asn-Leu-Asn-Glu-Glu-Gln-Arg-Asn-Ala-Lys-Ile-Lys-Ser-Ile-Arg-Asp-Asp(SEQ ID NO:12);

Ala-Val-Arg-Asn-Gly-Phe-Asn-Met-Gln-Gln-Gln-Arg-Arg-Phe-Tyr-Glu-Ala-Leu-His-Asp-Pro-Asn-Leu-Asn-Glu-Glu-Gln-Arg-Asn-Ala-Lys-Ile-Lys-Ser-Ile-Arg-Asp-Asp(SEQ ID NO:13);

Ala-Val-Ala-Gln-Ser-Phe-Asn-Met-Gln-Gln-Gln-Arg-Arg-Phe-Tyr-Glu-Ala-Leu-His-Asp-Pro-Asn-Leu-Asn-Glu-Glu-Gln-Arg-Asn-Ala-Lys-Ile-Lys-Ser-Ile-Arg-Asp-Asp(SEQ ID NO:14); and

Phe-Asn-Met-Gln-Gln-Gln-Arg-Arg-Phe-Tyr-Glu-Ala-Leu-His-Asp-Pro-Asn-Leu-Asn-Glu-Glu-Gln-Arg-Asn-Ala-Lys-Ile-Lys-Ser-Ile-Arg-Asp-Asp(SEQ ID NO: 15).

In an additional aspect, the invention provides a compound selected fromthe group consisting of

Ala-Val-Asp-Asn-Lys-Phe-Asn-Lys-Glu-Gln-Gln-Ala-Arg-Phe-Tyr-Glu-Ala-Leu-His-Asp-Pro-Asn-Leu-Asn-Glu-Glu-Gln-Arg-Asn-Ala-Lys-Ile-Gln-Ser-Ile-Lys-Asp-Asp(SEQ ID NO:16);

Ala-Val-Arg-Asn-Gly-Phe-Asn-Lys-Glu-Gln-Gln-Ala-Arg-Phe-Tyr-Glu-Ala-Leu-His-Asp-Pro-Asn-Leu-Asn-Glu-Glu-Gln-Arg-Asn-Ala-Lys-Ile-Gln-Ser-Ile-Lys-Asp-Asp(SEQ ID NO:17);

Ala-Val-Ala-Gln-Ser-Phe-Asn-Lys-Glu-Gln-Gln-Ala-Arg-Phe-Tyr-Glu-Ala-Leu-His-Asp-Pro-Asn-Leu-Asn-Glu-Glu-Gln-Arg-Asn-Ala-Lys-Ile-Gln-Ser-Ile-Lys-Asp-Asp(SEQ ID NO:18);

Ala-Val-Asp-Asn-Lys-Phe-Asn-Met-Gln-Gln-Gln-Ala-Arg-Phe-Tyr-Glu-Ala-Leu-His-Asp-Pro-Asn-Leu-Asn-Glu-Glu-Gln-Arg-Asn-Ala-Lys-Ile-Gln-Ser-Ile-Lys-Asp-Asp(SEQ ID NO: 19);

Ala-Val-Asp-Asn-Lys-Gly-Trp-Met-Arg-Gln-Gln-Ala-Arg-Phe-Tyr-Glu-Ala-Leu-His-Asp-Pro-Asn-Leu-Asn-Glu-Glu-Gln-Arg-Asn-Ala-Lys-Ile-Gln-Ser-Ile-Lys-Asp-Asp(SEQ ID NO:20);

Ala-Val-Asp-Asn-Lys-Phe-Asn-Lys-Glu-Gln-Gln-Ala-Ala-Phe-Tyr-Glu-Ala-Leu-His-Asp-Pro-Asn-Leu-Asn-Glu-Glu-Gln-Arg-Asn-Ala-Lys-Ile-Gln-Ser-Ile-Lys-Asp-Asp(SEQ ID NO:21);

Ala-Val-Asp-Asn-Lys-Phe-Asn-Lys-Glu-Gln-Gln-Ala-Arg-Phe-Tyr-Glu-Ala-Leu-His-Asp-Pro-Asn-Leu-Asn-Glu-Glu-Gln-Arg-Asn-Ala-Lys-Ile-Lys-Ser-Ile-Arg-Asp-Asp(SEQ ID NO:22);

Ala-Val-Arg-Asn-Gly-Phe-Asn-Met-Gln-Gln-Gln-Ala-Arg-Phe-Tyr-Glu-Ala-Leu-His-Asp-Pro-Asn-Leu-Asn-Glu-Glu-Gln-Arg-Asn-Ala-Lys-Ile-Lys-Ser-Ile-Arg-Asp-Asp(SEQ ID NO:23);

Ala-Val-Ala-Gln-Ser-Phe-Asn-Met-Gln-Gln-Gln-Ala-Arg-Phe-Tyr-Glu-Ala-Leu-His-Asp-Pro-Asn-Leu-Asn-Glu-Glu-Gln-Arg-Asn-Ala-Lys-Ile-Lys-Ser-Ile-Arg-Asp-Asp(SEQ ID NO:24); and

Phe-Asn-Met-Gln-Gln-Gln-Ala-Arg-Phe-Tyr-Glu-Ala-Leu-His-Asp-Pro-Asn-Leu-Asn-Glu-Glu-Gln-Arg-Asn-Ala-Lys-Ile-Lys-Ser-Ile-Arg-Asp-Asp(SEQ ID NO:25).

In a preferred embodiment, the invention provides peptides of Formula(Ia) wherein X₁ is Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ is Ala, AA₄ isGln, and AA₅ is Ser.

In another preferred embodiment, the invention provides peptides ofFormula (Ia) wherein X₁ is Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ isAla, AA₄ is Gln, AA₅ is Ser, AA₆ is Phe, AA₇ is Asn, AA₈ is Lys, AA₉ isGlu, AA₁₂ is Asn, AA₁₃ is Arg, AA₃₃ is Gln, and AA₃₆ is Lys.

In yet another aspect, the invention provides peptides of Formula (Ia)wherein X₁ is Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ is Ala, AA₄ is Gln,AA₅ is Ser, and AA₁₂ is Ala.

In still another aspect, the invention provides peptides of Formula (Ia)wherein X₁ is Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ is Ala, AA₄ is Gln,AA₅ is Ser, AA₆ is Phe, AA₇ is Asn, AA₈ is Lys, AA₉ is Glu, AA₁₂ is Ala,AA₁₃ is Arg, AA₃₃ is Gln, and AA₃₆ is Lys.

In yet another preferred embodiment, the invention provides peptides ofFormula (Ia) wherein AA₆ is Gly, AA₇ is Trp, AA₈ is Met, and AA₉ is Arg.

In still another preferred embodiment, the invention provides peptidesof Formula (Ia) wherein X₁ is Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₆ isGly, AA₇ is Trp, AA₈ is Met, AA₉ is Arg, AA₃ is Asp, AA₄ is Asn, AA₅ isLys, AA₂ is Asn, AA₁₃ is Arg, AA₃₃ is Gln; and AA₃₆ is Lys.

Also encompassed herein are peptides of Formula (Ia) wherein AA₆ is Gly,AA₇ is Trp, AA₈ is Met, AA₉ is Arg, and AA₁₂ is Ala.

Further encompassed herein are peptides of Formula (Ia) wherein X₁ isAla-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₆ is Gly, AA₇ is Trp, AA₈ is Met,AA₉ is Arg, AA₃ is Asp, AA₄ is Asn, AA₅ is Lys, AA₁₂ is Ala, AA₁₃ isArg, AA₃₃ is Gln; and AA₃₆ is Lys.

In a further preferred embodiment, the invention provides peptides ofFormula (Ia) wherein AA₁₂ is Arg.

Also preferred are peptides of Formula (Ia) wherein X₁ isAla-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₈ is Lys, AA₉ is Glu, AA₁₂ is Arg,AA₃ is Asp, AA₄ is Asn, AA₅ is Lys, AA₆ is Phe, AA₇is Asn, AA₁₃ is Arg,AA₃₃ is Gln, and AA₃₆is Lys.

Further encompassed within the scope of the invention are peptides ofFormula (Ia) wherein AA₁₂ is Ala.

Additionally encompassed within the scope of the invention are peptidesof Formula (Ia) wherein X₁ is Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₈ isLys, AA₉ is Glu, AA₁₂ is Ala, AA₃ is Asp, AA₄ is Asn, AA₅ is Lys, AA₆ isPhe, AA₇ is Asn, AA₁₃ is Arg, AA₃₃ is Gln, and AA₃₆ is Lys.

In an additional preferred embodiment, the invention provides peptidesof Formula (Ia) wherein AA₃₃ is Lys and AA₃₆ is Arg.

In still another preferred embodiment, the invention provides peptidesof Formula (Ia) wherein X₁ is Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃₃ isLys, AA₃₆ is Arg, AA₃ is Asp, AA₄ is Asn, AA₅ is Lys, AA₆ is Phe, AA₇ isAsn, AA₈ is Lys, AA₉ is Glu, AA₁₂ is Asn, and AA₁₃ is Arg.

Further encompassed herein are peptides of Formula (Ia) wherein AA₃₃ isLys, AA₃₆ is Arg, and AA₁₂ is Ala.

Additionally encompassed herein are peptides of Formula (Ia) wherein X₁is Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃₃ is Lys, AA₃₆ is Arg, AA₃ isAsp, AA₄ is Asn, AA₅ is Lys, AA₆ is Phe, AA₇ is Asn, AA₈ is Lys, AA₉ isGlu, AA₁₂ is Ala, and AA₁₃ is Arg.

In yet another preferred embodiment, the invention provides peptides ofFormula (Ia) wherein X₁ is Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ isArg, AA₅ is Gly, AA₈ is Met, AA₉ is Gln, AA₁₂ is Arg, AA₃₃ is Lys, andAA₃₆ is Arg.

In a more preferred embodiment, the invention provides peptides ofFormula (Ia) wherein X₁ is Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ isArg, AA₅ is Gly, AA₈ is Met, AA₉ is Gln, AA₁₂ is Arg, AA₃₃ is Lys, AA₃₆is Arg, AA₄ is Asn, AA₆ is Phe, AA₇ is Asn, and AA₁₃ is Arg.

Also provided herein are peptides of Formula (Ia) wherein X₁ isAla-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ is Arg, AA₅ is Gly, AA₈ is Met,AA₉ is Gln, AA₁₂ is Ala, AA₃₃ is Lys, and AA₃₆ is Arg.

Further provided herein are peptides of Formula (Ia) wherein X₁ isAla-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ is Arg, AA₅ is Gly, AA₈ is Met,AA₉ is Gln, AA₁₂ is Ala, AA₃₃ is Lys, AA₃₆ is Arg, AA₄ is Asn, AA₆ isPhe, AA₇ is Asn, and AA₃ is Arg.

In an additional preferred embodiment, the invention provides peptidesof Formula (Ia) wherein X₁ is Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ isAla, AA₄ is Gln, AA₅ is Ser, AA₈ is Met, AA₉ is Gln, AA₁₂ is Arg, AA₃₃is Lys, and AA₃₆ is Arg.

In a more preferred embodiment, the invention provides peptides ofFormula (Ia) wherein X₁ is Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ isAla, AA₄ is Gln, AA₅ is Ser, AA₈ is Met, AA₉ is Gln, AA₁₂ is Arg, AA₃₃is Lys, AA₃₆ is Arg, AA₆ is Phe, AA₇ is Asn, and AA₁₃ is Arg.

Additionally provided herein are peptides of Formula (Ia) wherein X₁ isAla-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ is Ala, AA₄ is Gln, AA₅ is Ser,AA₈ is Met, AA₉ is Gln, AA₁₂ is Ala, AA₃₃ is Lys, and AA₃₆ is Arg.

In another embodiment, the invention provides peptides of Formula (Ia)wherein X₁ is Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ is Ala, AA₄ is Gln,AA₅ is Ser, AA₈ is Met, AA₉ is Gln, AA₁₂ is Ala, AA₃₃ is Lys, AA₃₆ isArg, AA₆ is Phe, AA₇ is Asn, and AA₁₃ is Arg.

In a particularly preferred embodiment, the invention provides peptidesof Formula (Ia) wherein X₁ is absent, AA₈ is Met, AA₉ is Gln, AA₁₂ isArg, AA₃₃ is Lys, and AA₃₆ is Arg.

In an even more preferred embodiment, the invention provides peptides ofFormula (Ia) wherein X₁ is absent, AA₈ is Met, AA₉ is Gln, AA₁₂ is Arg,AA₃₃ is Lys, AA₃₆ is Arg, AA₆ is Phe, AA₇ is Asn, and AA₁₃ is Arg.

In yet another embodiment, the invention provides peptides of Formula(Ia) wherein X₁ is absent, AA₈ is Met, AA₉ is Gln, AA₁₂ is Ala, AA₃₃ isLys, and AA₃₆ is Arg.

In still another embodiment, the invention provides peptides of Formula(Ia) wherein X₁ is absent, AA₈ is Met, AA₉ is Gln, AA₁₂ is Ala, AA₃₃ isLys, AA₃₆ is Arg, AA₆ is Phe, AA₇is Asn, and AA₁₃ is Arg.

In another aspect, the invention provides Z domain variant peptides thatare conformationally restrained by an intramolecular disulfide bond inorder to improve the structural stability of the variant peptides. Itwas discovered that the substitution of a cysteine residue at amino acidposition 10 and the addition of a cysteine at the C-terminus of the Zdomain variants described herein followed by cyclization of suchpeptides results in the formation of variants which mimic the nativehelicity of the full-length, wild type Z domain. The cyclization of theZ domain variant peptides was also found to increase the IgG-bindingactivity of the variant peptides. Accordingly, the invention providescyclized Z domain variant peptides represented by Formula (II):

where

X₁ is selected from the group consisting of H, C₁-C₆ alkanoyl, andZ-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2);

where

Z is selected from the group consisting of H and C₁-C₆ alkanoyl;

AA₃ is selected from the group consisting of Asp, Arg, and Ala;

AA₄ is selected from the group consisting of Asn and Gln; and

AA₅ is selected from the group consisting of Lys, Gly, and Ser;

AA₆ is selected from the group consisting of Phe and Gly;

AA₇ is selected from the group consisting of Asn and Trp;

AA₈ is selected from the group consisting of Lys and Met;

AA₉ is selected from the group consisting of Glu, Gln, and Arg;

AA₁₂ is selected from the group consisting of Asn, Ala, and Arg;

AA₁₃ is selected from the group consisting of Ala and Arg;

AA₃₃ is selected from the group consisting of Gln and Lys;

AA₃₆ is selected from the group consisting of Lys and Arg; and

X₂ is selected from the group consisting of OR₁ and NR₁R₂ where R₁ andR₂ are independently selected from the group consisting of H,C₁-C₆alkyl, C₆-C₁₂aryl and C₆-C₁₂aryl-C₁-C₆alkyl.

In one embodiment, the invention provides peptides of Formula (II)wherein X₁ is Z-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ is Arg, and AA₅is Gly.

In another embodiment, the invention provides peptides of Formula (II)wherein X₁ is Z-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ is Arg, AA₅ isGly, AA₄ is Asn, AA₆ is Phe, AA₇ is Asn, AA₈ is Lys, AA₉ is Glu, AA₁₂ isAsn, AA₁₃ is Arg, AA₃₃ is Gln, and AA₃₆ is Lys.

In yet another embodiment, the invention provides peptides of Formula(II) wherein X₁ is Z-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ is Arg, AA₅is Gly, and AA₁₂ is Ala.

Further provided herein are peptides of Formula (II) wherein X₁ isZ-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ is Arg, AA₅ is Gly, AA₄ is Asn,AA₆ is Phe, AA₇ is Asn, AA₈ is Lys, AA₉ is Glu, AA₁₂ is Ala, AA₁₃ isArg, AA₃₃ is Gln, and AA₃₆ is Lys.

In still another embodiment, the invention provides peptides of Formula(II) wherein AA₈ is Met and AA₉ is Gln.

Also provided herein are peptides of Formula (II) wherein AA₈ is Met,AA₉ is Gln, X₁ is Z-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ is Asp, AA₄is Asn, AA₅ is Lys, AA₆ is Phe, AA₇ is Asn, AA₁₂ is Asn, AA₁₃ is Arg,AA₃₃ is Gln, and AA₃₆ is Lys.

Additionally provided herein are peptides of Formula (II) whereinAA_(8 is) Met, AA₉ is Gln, and AA₁₂ is Ala.

In a further aspect, the invention provides peptides of Formula (II)wherein AA₈ is Met, AA₉ is Gln, X₁ is Z-Ala-Val-AA₃-AA₄-AA₅ (SEQ IDNO:2), AA₃ is Asp, AA₄ is Asn, AA₅ is Lys, AA₆ is Phe, AA₇ is Asn, AA₁₂is Ala, AA₁₃ is Arg, AA₃₃ is Gln, and AA₃₆ is Lys.

In an additional embodiment, the invention provides peptides of Formula(II) wherein AA₁₂ is Arg and AA₁₃ is Ala.

In a further embodiment, the invention provides peptides of Formula (II)wherein AA₁₂ is Arg, AA₁₃ is Ala, X₁ is Z-Ala-Val-AA₃-AA₄-AA₅ (SEQ IDNO:2), AA₃ is Asp, AA₄ is Asn, AA₅ is Lys, AA₆ is Phe, AA₇ is Asn, AA₈is Lys, AA₉ is Glu, AA₃₃ is Gln, and AA₃₆ is Lys.

Also encompassed within the scope of the invention are peptides ofFormula (II) wherein AA₁₂ is Ala and AA₁₃ is Ala.

Further encompassed within the scope of the invention are peptides ofFormula (II) wherein AA₁₂ is Ala, AA₁₃ is Ala, X₁ isZ-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ is Asp, AA₄ is Asn, AA₅ is Lys,AA₆ is Phe, AA₇ is Asn, AA₈ is Lys, AA₉ is Glu, AA₃₃ is Gln; and AA₃₆ isLys.

In yet another aspect, the invention provides a cyclized Z domainvariant peptide selected from the group consisting of

where

X₁ is selected from the group consisting of H and C₁-C₆alkanoyl;

Z is selected from the group consisting of H and C₁-C₆alkanoyl; and

X₂ is selected from the group consisting of OR₁ and NR₁R₂ where R₁ andR₂ are independently selected from the group consisting of H,C₁-C₆alkyl, C₆-C₁₂aryl and C₆-C₁₂aryl-C₁-C₆alkyl.

In still another aspect, the invention provides a cyclized Z domainvariant peptide selected from the group consisting of

where

X₁ is selected from the group consisting of H and C₁-C₆alkanoyl;

Z is selected from the group consisting of H and C₁-C₆ alkanoyl; and

X₂ is selected from the group consisting of OR₁ and NR₁R₂ where R₁ andR₂ are independently selected from the group consisting of H, C₁-C₆alkyl, C₆-C₁₂aryl and C₆-C₁₂aryl-C₁-C₆alkyl.

In a preferred embodiment, the invention provides peptides of Formula(II) wherein X₁ is Z-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ is Ala, AA₄is Gln, and AA₅ is Ser.

In another preferred embodiment, the invention provides peptides ofFormula (II) wherein X₁ is Z-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ isAla, AA₄ is Gln, AA₅ is Ser, AA₆ is Phe, AA₇ is Asn, AA₈ is Lys, AA₉ isGlu, AA₁₂ is Asn, AA₁₃ is Arg, AA₃₃ is Gln, and AA₃₆ is Lys.

In yet another aspect, the invention provides peptides of Formula (II)wherein X₁ is Z-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ is Ala, AA₄ isGln, AA₅ is Ser, and AA₁₂ is Ala.

In still another aspect, the invention provides peptides of Formula (II)wherein X₁ is Z-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ is Ala, AA₄ isGln, AA₅ is Ser, AA₆ is Phe, AA₇ is Asn, AA₈ is Lys, AA₉ is Glu, AA₁₂ isAla, AA₁₃ is Arg, AA₃₃ is Gln, and AA₃₆ is Lys.

In yet another preferred embodiment, the invention provides peptides ofFormula (II) wherein AA₆ is Gly, AA₇ is Trp, AA₈ is Met, and AA₉ is Arg.

In still another preferred embodiment, the invention provides peptidesof Formula (II) wherein X₁ is Z-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₆is Gly, AA₇ is Trp, AA₈ is Met, AA₉ is Arg, AA₃ is Asp, AA₄ is Asn, AA₅is Lys, AA₁₂ is Asn, AA₁₃ is Arg, AA₃₃ is Gln; and AA₃₆ is Lys.

Also encompassed herein are peptides of Formula (II) wherein AA₆ is Gly,AA₇ is Trp, AA₈ is Met, AA₉ is Arg, and AA₁₂ is Ala.

Further encompassed herein are peptides of Formula (II) wherein X₁ isZ-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₆ is Gly, AA₇ is Trp, AA₈ is Met,AA₉ is Arg, AA₃ is Asp, AA₄ is Asn, AA₅ is Lys, AA₁₂ is Ala, AA₁₃ isArg, AA₃₃ is Gln; and AA₃₆ is Lys.

In a further preferred embodiment, the invention provides peptides ofFormula (II) wherein AA₁₂ is Arg.

Also preferred are peptides of Formula (II) wherein X₁ isZ-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₁₂ is Arg, AA₃ is Asp, AA₄ isAsn, AA₅ is Lys, AA₆ is Phe, AA₇ is Asn, AA₈ is Lys, AA₉ is Glu, AA₁₃ isArg, AA₃₃ is Gln, and AA₃₆ is Lys.

Further encompassed within the scope of the invention are peptides ofFormula (II) wherein AA₁₂ is Ala.

Additionally encompassed within the scope of the invention are peptidesof Formula (II) wherein X₁ is Z-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₁₂is Ala, AA₃ is Asp, AA₄ is Asn, AA₅ is Lys, AA₆ is Phe, AA₇ is Asn, AA₈is Lys, AA₉ is Glu, AA₁₃ is Arg, AA₃₃ is Gln, and AA₃₆ is Lys.

In an additional preferred embodiment, the invention provides peptidesof Formula (II) wherein AA₃₃ is Lys and AA₃₆ is Arg.

In still another preferred embodiment, the invention provides peptidesof Formula (II) wherein X₁ is Z-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃₃is Lys, AA₃₆ is Arg, AA₃ is Asp, AA₄ is Asn, AA₅ is Lys, AA₆ is Phe, AA₇is Asn, AA₈ is Lys, AA₉ is Glu, AA₁₂ is Asn, and AA₁₃ is Arg.

Further encompassed herein are peptides of Formula (II) wherein AA₃₃ isLys, AA₃₆ is Arg, and AA₁₂ is Ala.

Additionally encompassed herein are peptides of Formula (II) wherein X₁is Z-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃₃ is Lys, AA₃₆ is Arg, AA₃ isAsp, AA₄ is Asn, AA₅ is Lys, AA₆ is Phe, AA₇ is Asn, AA₈ is Lys, AA₉ isGlu, AA₁₂ is Ala, and AA₁₃ is Arg.

In yet another preferred embodiment, the invention provides peptides ofFormula (II) wherein X₁ is Z-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ isArg, AA₅ is Gly, AA₈ is Met, AA₉ is Gln, AA₁₂ is Arg, AA₃₃ is Lys, andAA₃₆ is Arg.

In a more preferred embodiment, the invention provides peptides ofFormula (II) wherein X₁ is Z-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ isArg, AA₅ is Gly, AA₈ is Met, AA₉ is Gln, AA₁₂ is Arg, AA₃₃ is Lys, AA₃₆is Arg, AA₄ is Asn, AA₆ is Phe, AA₇ is Asn, and AA₁₃ is Arg.

Also provided herein are peptides of Formula (II) wherein X₁ isZ-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ is Arg, AA₅ is Gly, AA₅ is Met,AA₉ is Gln, AA₁₂ is Ala, AA₃₃ is Lys, and AA₃₆ is Arg.

Further provided herein are peptides of Formula (II) wherein X₁ isZ-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ is Arg, AA₅ is Gly, AA₈ is Met,AA₉ is Gln, AA₁₂ is Ala, AA₃₃ is Lys, AA₃₆ is Arg, AA₄ is Asn, AA₆ isPhe, AA₇ is Asn, and AA₁₃ is Arg.

In an additional preferred embodiment, the invention provides peptidesof Formula (II) wherein X₁ is Z-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃is Ala, AA₄ is Gln, AA₅ is Ser, AA₈ is Met, AA₉ is Gln, AA₁₂ is Arg,AA₃₃ is Lys, and AA₃₆ is Arg.

In a more preferred embodiment, the invention provides peptides ofFormula (II) wherein X₁ is Z-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ isAla, AA₄ is Gln, AA₅ is Ser, AA₈ is Met, AA₉ is Gln, AA₁₂ is Arg, AA₃₃is Lys, AA₃₆ is Arg, AA₆ is Phe, AA₇ is Asn, and AA₁₃ is Arg.

Additionally provided herein are peptides of Formula (II) wherein X₁ isZ-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ is Ala, AA₄ is Gln, AA₅ is Ser,AA₈ is Met, AA₉ is Gln, AA₁₂ is Ala, AA₃₃ is Lys, and AA₃₆ is Arg.

In another embodiment, the invention provides peptides of Formula (II)wherein X₁ is Z-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ is Ala, AA₄ isGln, AA₅ is Ser, AA₈ is Met, AA₉ is Gln, AA₁₂ is Ala, AA₃₃ is Lys, AA₃₆is Arg, AA₆ is Phe, AA₇ is Asn, and AA₁₃ is Arg.

In a particularly preferred embodiment, the invention provides peptidesof Formula (II) wherein X₁ is H or C₁-C₆ alkanoyl, AA₈ is Met, AA₉ isGln, AA₁₂ is Arg, AA₃₃ is Lys, and AA₃₆ is Arg.

In an even more preferred embodiment, the invention provides peptides ofFormula (II) wherein X₁ is H or C₁-C₆ alkanoyl, AA₈ is Met, AA₉ is Gln,AA₁₂ is Arg, AA₃₃ is Lys, AA₃₆ is Arg, AA₆ is Phe, AA₇ is Asn, and AA₁₃is Arg.

In yet another embodiment, the invention provides peptides of Formula(II) wherein X₁ is H or C₁-C₆ alkanoyl, AA₈ is Met, AA₉ is Gln, AA₁₂ isAla, AA₃₃ is Lys, and AA₃₆ is Arg.

In still another embodiment, the invention provides peptides of Formula(II) wherein X₁ is H or C₁-C₆ alkanoyl, AA₈ is Met, AA₉ is Gln, AA₁₂ isAla, AA₃₃ is Lys, AA₃₆ is Arg, AA₆ is Phe, AA₇ is Asn, and AA₁₃ is Arg.

In another embodiment, the invention provides Z domain variant peptidesrepresented by Formula (IIa):

where

X₁ is absent or is Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2);

where

AA₃ is selected from the group consisting of Asp, Arg, and Ala;

AA₄ is selected from the group consisting of Asn and Gln; and

AA₅ is selected from the group consisting of Lys, Gly, and Ser;

AA₆ is selected from the group consisting of Phe and Gly;

AA₇ is selected from the group consisting of Asn and Trp;

AA₈ is selected from the group consisting of Lys and Met;

AA₉ is selected from the group consisting of Glu, Gln, and Arg;

AA₁₂ is selected from the group consisting of Asn, Ala, and Arg;

AA₁₃ is selected from the group consisting of Ala and Arg;

AA₃₃ is selected from the group consisting of Gln and Lys; and

AA₃₆ is selected from the group consisting of Lys and Arg.

In one embodiment, the invention provides peptides of Formula (IIa)wherein X₁ is Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ is Arg, and AA₅ isGly.

In another embodiment, the invention provides peptides of Formula (IIa)wherein X₁ is Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ is Arg, AA₅ is Gly,AA₄ is Asn, AA₆ is Phe, AA₇ is Asn, AA₈ is Lys, AA₉ is Glu, AA₁₂ is Asn,AA₁₃ is Arg, AA₃₃ is Gln, and AA₃₆ is Lys.

In yet another embodiment, the invention provides peptides of Formula(IIa) wherein X₁ is Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ is Arg, AA₅is Gly, and AA₁₂ is Ala.

Further provided herein are peptides of Formula (IIa) wherein X₁ isAla-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ is Arg, AA₅ is Gly, AA₄ is Asn,AA₆ is Phe, AA₇ is Asn, AA₈ is Lys, AA₉ is Glu, AA₁₂ is Ala, AA₁₃ isArg, AA₃₃ is Gln, and AA₃₆ is Lys.

In still another embodiment, the invention provides peptides of Formula(IIa) wherein AA₈ is Met and AA₉ is Gln.

Also provided herein are peptides of Formula (IIa) wherein AA₈ is Met,AA₉ is Gln, X₁ is Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ is Asp, AA₄ isAsn, AA₅ is Lys, AA₆ is Phe, AA₇ is Asn, AA₁₂ is Asn, AA₁₃ is Arg, AA₃₃is Gln, and AA₃₆ is Lys.

Additionally provided herein are peptides of Formula (IIa) wherein AA₈is Met, AA₉ is Gln, and AA₁₂ is Ala.

In a further aspect, the invention provides peptides of Formula (IIa)wherein AA₈ is Met, AA₉ is Gln, X₁ is Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2),AA₃ is Asp, AA₄ is Asn, AA₅ is Lys, AA₆ is Phe, AA₇ is Asn, AA₁₂ is Ala,AA₁₃ is Arg, AA₃₃ is Gln, and AA₃₆ is Lys.

In an additional embodiment, the invention provides peptides of Formula(IIa) wherein AA₁₂ is Arg and AA₁₃ is Ala.

In a further embodiment, the invention provides peptides of Formula(IIa) wherein AA₁₂ is Arg, AA₁₃ is Ala, X₁ is Ala-Val-AA₃-AA₄-AA₅ (SEQID NO:2), AA₃ is Asp, AA₄ is Asn, AA₅ is Lys, AA₆ is Phe, AA₇ is Asn,AA₈ is Lys, AA₉ is Glu, AA₃₃ is Gln, and AA₃₆ is Lys.

Also encompassed within the scope of the invention are peptides ofFormula (IIa) wherein AA₁₂ is Ala and AA₁₃ is Ala.

Further encompassed within the scope of the invention are peptides ofFormula (IIa) wherein AA₁₂ is Ala, AA₁₃ is Ala, X₁ isAla-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ is Asp, AA₄ is Asn, AA₅ is Lys,AA₆ is Phe, AA₇ is Asn, AA₈ is Lys, AA₉ is Glu, AA₃₃ is Gln; and AA₃₆ isLys.

In yet another aspect, the invention provides a cyclized Z domainvariant peptide selected from the group consisting of

In still another aspect, the invention provides a cyclized Z domainvariant peptide selected from the group consisting of

In a preferred embodiment, the invention provides peptides of Formula(IIa) wherein X₁ is Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ is Ala, AA₄is Gln, and AA₅ is Ser.

In another preferred embodiment, the invention provides peptides ofFormula (IIa) wherein X₁ is Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ isAla, AA₄ is Gln, AA₅ is Ser, AA₆ is Phe, AA₇ is Asn, AA₈ is Lys, AA₉ isGlu, AA₁₂ is Asn, AA₁₃ is Arg, AA₃₃ is Gln, and AA₃₆ is Lys.

In yet another aspect, the invention provides peptides of Formula (IIa)wherein X₁ is Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ is Ala, AA₄ is Gln,AA₅ is Ser, and AA₁₂ is Ala.

In still another aspect, the invention provides peptides of Formula(IIa) wherein X₁ is Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ is Ala, AA₄is Gln, AA₅ is Ser, AA₆ is Phe, AA₇ is Asn, AA₈ is Lys, AA₉ is Glu, AA₁₂is Ala, AA₁₃ is Arg, AA₃₃ is Gln, and AA₃₆ is Lys.

In yet another preferred embodiment, the invention provides peptides ofFormula (IIa) wherein AA₆ is Gly, AA₇ is Trp, AA₈ is Met, and AA₉ isArg.

In still another preferred embodiment, the invention provides peptidesof Formula (IIa) wherein X₁ is Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₆ isGly, AA₇ is Trp, AA₈ is Met, AA₉ is Arg, AA₃ is Asp, AA₄ is Asn, AA₅ isLys, AA₁₂ is Asn, AA₁₃ is Arg, AA₃₃ is Gln; and AA₃₆ is Lys.

Also encompassed herein are peptides of Formula (IIa) wherein AA₆ isGly, AA₇ is Trp, AA₈ is Met, AA₉ is Arg, and AA₁₂ is Ala.

Further encompassed herein are peptides of Formula (IIa) wherein X₁ isAla-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₆ is Gly, AA₇ is Trp, AA₈ is Met,AA₉ is Arg, AA₃ is Asp, AA₄ is Asn, AA₅ is Lys, AA₁₂ is Ala, AA₁₃ isArg, AA₃₃ is Gln; and AA₃₆ is Lys.

In a further preferred embodiment, the invention provides peptides ofFormula (IIa) wherein AA₁₂ is Arg.

Also preferred are peptides of Formula (IIa) wherein X₁ isAla-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₁₂ is Arg, AA₃ is Asp, AA₄ is Asn,AA₅ is Lys, AA₆ is Phe, AA₇ is Asn, AA₈ is Lys, AA₉ is Glu, AA₁₃ is Arg,AA₃₃ is Gln, and AA₃₆ is Lys.

Further encompassed within the scope of the invention are peptides ofFormula (IIa) wherein AA₁₂ is Ala.

Additionally encompassed within the scope of the invention are peptidesof Formula (IIa) wherein X₁ is Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₁₂is Ala, AA₃ is Asp, AA₄ is Asn, AA₅ is Lys, AA₆ is Phe, AA₇ is Asn, AA₈is Lys, AA₉ is Glu, AA₁₃ is Arg, AA₃₃ is Gln, and AA₃₆ is Lys.

In an additional preferred embodiment, the invention provides peptidesof Formula (IIa) wherein AA₃₃ is Lys and AA₃₆ is Arg.

In still another preferred embodiment, the invention provides peptidesof Formula (IIa) wherein X₁ is Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃₃is Lys, AA₃₆ is Arg, AA₃ is Asp, AA₄ is Asn, AA₅ is Lys, AA₆ is Phe, AA₇is Asn, AA₈ is Lys, AA₉ is Glu, AA₁₂ is Asn, and AA₁₃ is Arg.

Further encompassed herein are peptides of Formula (IIa) wherein AA₃₃ isLys, AA₃₆ is Arg, and AA₁₂ is Ala.

Additionally encompassed herein are peptides of Formula (IIa) wherein X₁is Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃₃ is Lys, AA₃₆ is Arg, AA₃ isAsp, AA₄ is Asn, AA₅ is Lys, AA₆ is Phe, AA₇ is Asn, AA₈ is Lys, AA₉ isGlu, AA₁₂ is Ala, and AA₁₃ is Arg.

In yet another preferred embodiment, the invention provides peptides ofFormula (IIa) wherein X₁ is Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ isArg, AA₅ is Gly, AA₈ is Met, AA₉ is Gln, AA₁₂ is Arg, AA₃₃ is Lys, andAA₃₆ is Arg.

In a more preferred embodiment, the invention provides peptides ofFormula (IIa) wherein X₁ is Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ isArg, AA₅ is Gly, AA₈ is Met, AA₉ is Gln, AA₁₂ is Arg, AA₃₃ is Lys, AA₃₆is Arg, AA₄ is Asn, AA₆ is Phe, AA₇ is Asn, and AA₁₃ is Arg.

Also provided herein are peptides of Formula (IIa) wherein X₁ isAla-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ is Arg, AA₅ is Gly, AA₈ is Met,AA₉ is Gln, AA₁₂ is Ala, AA₃₃ is Lys, and AA₃₆ is Arg.

Further provided herein are peptides of Formula (IIa) wherein X₁ isAla-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ is Arg, AA₅ is Gly, AA₈ is Met,AA₉ is Gln, AA₁₂ is Ala, AA₃₃ is Lys, AA₃₆ is Arg, AA₄ is Asn, AA₆ isPhe, AA₇ is Asn, and AA₃ is Arg.

In an additional preferred embodiment, the invention provides peptidesof Formula (IIa) wherein X₁ is Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ isAla, AA₄ is Gln, AA₅ is Ser, AA₈ is Met, AA₉ is Gln, AA₁₂ is Arg, AA₃₃is Lys, and AA₃₆ is Arg.

In a more preferred embodiment, the invention provides peptides ofFormula (IIa) wherein X₁ is Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ isAla, AA₄ is Gln, AA₅ is Ser, AA₈ is Met, AA₉ is Gln, AA₁₂ is Arg, AA₃₃is Lys, AA₃₆ is Arg, AA₆ is Phe, AA₇ is Asn, and AA₁₃ is Arg.

Additionally provided herein are peptides of Formula (IIa) wherein X₁ isAla-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ is Ala, AA₄ is Gln, AA₅ is Ser,AA₈ is Met, AA₉ is Gln, AA₁₂ is Ala, AA₃₃ is Lys, and AA₃₆ is Arg.

In another embodiment, the invention provides peptides of Formula (IIa)wherein X₁ is Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2), AA₃ is Ala, AA₄ is Gln,AA₅ is Ser, AA₈ is Met, AA₉ is Gln, AA₁₂ is Ala, AA₃₃ is Lys, AA₃₆ isArg, AA₆ is Phe, AA₇ is Asn, and AA₁₃ is Arg.

In a particularly preferred embodiment, the invention provides peptidesof Formula (IIa) wherein X₁ is absent, AA₈ is Met, AA₉ is Gln, AA₁₂ isArg, AA₃₃ is Lys, and AA₃₆ is Arg.

In an even more preferred embodiment, the invention provides peptides ofFormula (IIa) wherein X₁ is absent, AA₈ is Met, AA₉ is Gln, AA₁₂ is Arg,AA₃₃ is Lys, AA₃₆ is Arg, AA₆ is Phe, AA₇ is Asn, and AA₁₃ is Arg.

In yet another embodiment, the invention provides peptides of Formula(IIa) wherein X₁ is absent, AA₈ is Met, AA₉ is Gln, AA₁₂ is Ala, AA₃₃ isLys, and AA₃₆ is Arg.

In still another embodiment, the invention provides peptides of Formula(IIa) wherein X₁ is absent, AA₈ is Met, AA₉ is Gln, AA₁₂ is Ala, AA₃₃ isLys, AA₃₆ is Arg, AA₆ is Phe, AA₇ is Asn, and AA₁₃ is Arg.

In a preferred embodiment, the Z domain variant peptides of theinvention are non-covalently adsorbed or covalently bound to amacromolecule, such as a solid support. It will be appreciated that theinvention encompasses both macromolecules complexed with the cyclized Zdomain variant peptides provided herein and macromolecules complexedwith the linear Z domain variant peptides provided herein. In general,the solid support is an inert matrix, such as a polymeric gel,comprising a three dimensional structure, lattice or network of amaterial. Almost any macromolecule, synthetic or natural, can form a gelin a suitable liquid when suitably cross-linked with a bifunctionalreagent. Preferably, the macromolecule selected is convenient for use inaffinity chromatography. Most chromatographic matrices used for affinitychromatography are xerogels. Such gels shrink on drying to a compactsolid comprising only the gel matrix. When the dried xerogel isresuspended in the liquid, the gel matrix imbibes liquid, swells andreturns to the gel state. Xerogels suitable for use herein includepolymeric gels, such as cellulose, cross-linked dextrans (e.g.Sepharose), agarose, cross-linked agarose, polyacrylamide gels, andpolyacrylamide-agarose gels.

Alternatively, aerogels can be used for affinity chromatography. Thesegels do not shrink on drying but merely allow penetration of thesurrounding air. When the dry gel is exposed to liquid, the latterdisplaces the air in the gel. Aerogels suitable for use herein includeporous glass and ceramic gels.

Also encompassed herein are the Z domain variant peptides of theinvention coupled to derivatized gels wherein the derivative moietiesfacilitate the coupling of the peptide ligands to the gel matrix andavoid steric hindrance of the peptide ligand-IgG interaction in affinitychromatography. Alternatively, spacer arms can be interposed between thegel matrix and the peptide ligand for similar benefits.

In another embodiment, the invention provides fusion proteins in which aselected or desired polypeptide is fused at its N-terminus or itsC-terminus, or at both terminii, to one or more of the present Z domainvariant peptides. The invention contemplates the use of both cyclized Zdomain variant peptides and linear Z domain variant peptides in fusionproteins. In a preferred embodiment, the fusion protein is specificallycleavable such that at least a substantial portion of the Z domainvariant peptide sequence can be proteolytically cleaved away from thefusion protein to yield the desired polypeptide. The fusion proteins ofthe invention can be designed with cleavage sites recognized by chemicalor enzymatic proteases. In one embodiment, the fusion protein isdesigned with a unique cleavage site (or sites) for removal of the Zdomain variant peptide sequence, i.e. the fusion protein is designedsuch that a given protease (or proteases) cleaves away the Z domainvariant peptide sequence but does not cleave at any site within thesequence of the desired protein, avoiding fragmentation of the desiredprotein. In another embodiment, the cleavage site (or sites) at thefusion joint (or joints) is designed such that cleavage of the fusionprotein with a given enzyme liberates the authentic, intact sequence ofthe desired protein from the remainder of the fusion protein sequence.

II. Synthesis of Z Domain Variant Peptides 1. Chemical Synthesis a.General Procedures

One method of producing the Z domain variants of the invention involveschemical synthesis of the peptides. This can be accomplished by usingmethodologies well known in the art (see Kelley, R. F. & Winkler, M. E.in Genetic Engineering Principles and Methods, Setlow, J. K, ed., PlenumPress, N.Y., vol. 12, pp 1-19 (1990), Stewart, J. M. Young, J. D., SolidPhase Peptide Synthesis, Pierce Chemical Co., Rockford, Ill. (1984); seealso U.S. Pat. Nos. 4,105,603; 3,972,859; 3,842,067; and 3,862,925).

Peptides of the invention can be conveniently prepared using solid phasepeptide synthesis (Merrifield, J. Am. Chem. Soc., 85: 2149 (1964);Houghten, Proc. Natl. Acad. Sci. USA, 82: 5132 (1985). Solid phasesynthesis begins at the carboxy terminus of the putative peptide bycoupling a protected amino acid to an inert solid support. The inertsolid support can be any macromolecule capable of serving as an anchorfor the C-terminus of the initial amino acid. Typically, themacromolecular support is a cross-linked polymeric resin (e.g. apolyamide or polystyrene resin) as shown in FIGS. 1-1 and 1-2, on pages2 and 4 of Stewart and Young, supra. In one embodiment, the C-terminalamino acid is coupled to a polystyrene resin to form a benzyl ester. Amacromolecular support is selected such that the peptide anchor link isstable under the conditions used to deprotect the α-amino group of theblocked amino acids in peptide synthesis. If an base-labile α-protectinggroup is used, then it is desirable to use an acid-labile link betweenthe peptide and the solid support. For example, an acid-labile etherresin is effective for base-labile Fmoc-amino acid peptide synthesis asdescribed on page 16 of Stewart and Young, supra. Alternatively, apeptide anchor link and α-protecting group that are differentiallylabile to acidolysis can be used. For example, an aminomethyl resin suchas the phenylacetamidomethyl (Pam) resin works well in conjunction withBoc-amino acid peptide synthesis as described on pages 11-12 of Stewartand Young, supra.

After the initial amino acid is coupled to an inert solid support, theα-amino protecting group of the initial amino acid is removed with, forexample, trifluoroacetic acid (TFA) in methylene chloride andneutralizing in, for example, triethylamine (TEA). Followingdeprotection of the initial amino acid's α-amino group, the next α-aminoand sidechain protected amino acid in the synthesis is added. Theremaining α-amino and, if necessary, side chain protected amino acidsare then coupled sequentially in the desired order by condensation toobtain an intermediate compound connected to the solid support.Alternatively, some amino acids may be coupled to one another to form afragment of the desired peptide followed by addition of the peptidefragment to the growing solid phase peptide chain.

The condensation reaction between two amino acids, or an amino acid anda peptide, or a peptide and a peptide can be carried out according tothe usual condensation methods such as the axide method, mixed acidanhydride method, DCC (N,N′-dicyclohexylcarbodiimide) or DIC(N,N′-diisopropylcarbodiimide) methods, active ester method,p-nitrophenyl ester method, BOP (benzotriazole-1-yl-oxy-tris[dimethylamino] phosphonium hexafluorophosphate) method,N-hydroxysuccinic acid imido ester method, etc, and Woodward reagent Kmethod.

It is common in the chemical syntheses of peptides to protect anyreactive side-chain groups of the amino acids with suitable protectinggroups. Ultimately, these protecting groups are removed after thedesired polypeptide chain has been sequentially assembled. Also commonis the protection of the α-amino group on an amino acid or peptidefragment while the C-terminal carboxy group of the amino acid or peptidefragment reacts with the free N-terminal amino group of the growingsolid phase polypeptide chain, followed by the selective removal of theα-amino group to permit the addition of the next amino acid or peptidefragment to the solid phase polypeptide chain. Accordingly, it is commonin polypeptide synthesis that an intermediate compound is produced whichcontains each of the amino acid residues located in the desired sequencein the peptide chain wherein individual residues still carry side-chainprotecting groups. These protecting groups can be removed substantiallyat the same time to produce the desired polypeptide product followingremoval from the solid phase.

α- and ε-amino side chains can be protected with benzyloxycarbonyl(abbreviated Z), isonicotinyloxycarbonyl (iNOC),o-chlorobenzyloxycarbonyl [Z(2Cl)], p-nitrobenzyloxycarbonyl [Z(NO₂)],p-methoxybenzyloxycarbonyl [Z(OMe)], t-butoxycarbonyl (Boc),t-amyloxycarbonyl (Aoc), isobornyloxycarbonyl, adamantyloxycarbonyl,2-(4-biphenyl)-2-propyloxycarbonyl (Bpoc), 9-fluorenylmethoxycarbonyl(Fmoc), methylsulfonyethoxycarbonyl (Msc), trifluoroacetyl, phthalyl,formyl, 2-nitrophenylsulphenyl (NPS), diphenylphosphinothioyl (Ppt), anddimethylphosphinothioyl (Mpt) groups, and the like.

Protective groups for the carboxy functional group are exemplified bybenzyl ester (OBzl), cyclohexyl ester (Chx), 4-nitrobenzyl ester (ONb),t-butyl ester (Obut), 4-pyridylmethyl ester (OPic), and the like. It isoften desirable that specific amino acids such as arginine, cysteine,and serine possessing a functional group other than amino and carboxylgroups are protected by a suitable protective group. For example, theguanidino group of arginine may be protected with nitro,p-toluenesulfonyl, benzyloxycarbonyl, adamantyloxycarbonyl,p-methoxybenzesulfonyl, 4-methoxy-2,6-dimethylbenzenesulfonyl (Nds),1,3,5-trimethylphenysulfonyl (Mts), and the like. The thiol group ofcysteine can be protected with p-methoxybenzyl, trityl, and the like.

Many of the blocked amino acids described above can be obtained fromcommercial sources such as Novabiochem (San Diego, Calif.), Bachem, CA(Torrence, Calif.) or Peninsula Labs (Belmont, Calif.).

Stewart and Young, supra, provides detailed information regardingprocedures for preparing peptides. Protection of α-amino groups isdescribed on pages 14-18, and side chain blockage is described on pages18-28. A table of protecting groups for amine, hydroxyl and sulfhydrylfunctions is provided on pages 149-151.

After the desired amino acid sequence has been completed, the peptidecan be cleaved away from the solid support, recovered and purified. Thepeptide is removed from the solid support by a reagent capable ofdisrupting the peptide-solid phase link, and optionally deprotectsblocked side chain functional groups on the peptide. In one embodiment,the peptide is cleaved away from the solid phase by acidolysis withliquid hydrofluoric acid (HF), which also removes any remaining sidechain protective groups. Preferably, in order to avoid alkylation ofresidues in the peptide (for example, alkylation of methionine,cysteine, and tyrosine residues), the acidolysis reaction mixturecontains thio-cresol and cresol scavengers. Following HF cleavage, theresin is washed with ether, and the free peptide is extracted from thesolid phase with sequential washes of acetic acid solutions. Thecombined washes are lyophilized, and the peptide is purified.

In one embodiment, the peptides of the invention are synthesizedaccording to the method of Fields et al., Int. J. Peptide Protein Res.,35: 161-214 (1990), as described in Example 2 below.

b. Disulfide Linked Z Domain Variant Peptides

As described in Section B(I) above, some embodiments of the inventionprovide Z domain variant peptides that are cyclized by formation of adisulfide bond between cysteine residues substituted at amino acidpositions 10 and 39 of the Z domain template amino acid sequence. Suchpeptides can be made by chemical synthesis as described above and thencyclized by any convenient method used in the formation of disulfidelinkages. For example, peptides can be recovered from solid phasesynthesis with sulfhydryls in reduced form, dissolved in a dilutesolution wherein the intramolecular cysteine concentration exceeds theintermolecular cysteine concentration in order to optimizeintramolecular disulfide bond formation, such as a peptide concentrationof 25 mM to 1 μM, and preferably 500 μM to 1 μM, and more preferably 25μM to 1 μM, and then oxidized by exposing the free sulfhydryl groups toa mild oxidizing agent that is sufficient to generate intramoleculardisulfide bonds, e.g. molecular oxygen with or without catalysts such asmetal cations, potassium ferricyanide, sodium tetrathionate, etc. In oneembodiment, the peptides are cyclized as described in Example 2 below.Alternatively, the peptides can be cyclized as described in Pelton etal., J. Med Chem., 29: 2370-2375 (1986).

2. Recombinant Synthesis

In a further embodiment, the present invention encompasses a compositionof matter comprising isolated nucleic acid, preferably DNA, encoding apolypeptide selected from the group consisting of the Z domain variantpeptides described in Table 2 below. DNAs encoding the Z domain variantpeptides of the invention can be prepared by a variety of methods knownin the art. These methods include, but are not limited to, chemicalsynthesis by any of the methods described in Engels et al., Agnew. Chem.Int. Ed. Engl., 28: 716-734 (1989), the entire disclosure of which isincorporated herein by reference, such as the triester, phosphite,phosphoramidite and H-phosphonate methods. In one embodiment, codonspreferred by the expression host cell are used in the design of the Zdomain variant-encoding DNA. Alternatively, DNA encoding the wild type Zdomain can be isolated from a genomic or cDNA library, and the wild typeDNA sequence can be altered to encode one of the Z domain variants byusing recombinant DNA techniques, such as site specific mutagenesis(Kunkel et al., Methods Enzymol. 204:125-139 (1991); Carter, P., et al.,Nucl. Acids. Res. 13:4331 (1986); Zoller, M. J. et al., Nucl. Acids Res.10:6487 (1982)), cassette mutagenesis (Wells, J. A., et al., Gene 34:315(1985)), restriction selection mutagenesis (Wells, J. A., et al.,Philos. Trans, R. Soc. London SerA 317, 415 (1986)), and the like.

The invention further comprises an expression control sequence operablylinked to the DNA molecule encoding a Z domain variant peptide of Table2, and an expression vector, such as a plasmid, comprising the DNAmolecule, wherein the control sequence is recognized by a host celltransformed with the vector. In general, plasmid vectors containreplication and control sequences which are derived from speciescompatible with the host cell. The vector ordinarily carries areplication site, as well as sequences which encode proteins that arecapable of providing phenotypic selection in transformed cells.

For expression in prokaryotic hosts, suitable vectors include pBR322(ATCC No. 37,017), phGH107 (ATCC No. 40,011), pBO475, pS0132, pRIT5, anyvector in the pRIT20 or pRIT30 series (Nilsson and Abrahmsen, Meth.Enzymol., 185: 144-161 (1990)), pRIT2T, pKK233-2, pDR540 and pPL-lambda.Prokaryotic host cells containing the expression vectors of the presentinvention include E. coli K12 strain 294 (ATCC NO. 31446), E. colistrain JM101 (Messing et al., Nucl. Acid. Res., 9: 309 (1981)), E. colistrain B, E. coli strain₁₀₂ 1776 (ATCC No. 31537), E. coli c600(Appleyard, Genetics, 39: 440 (1954)), E. coli W3110 (F-, gamma-,prototrophic, ATCC No. 27325), E. coli strain 27C7 (W3110, tonA, phoAE15, (argF-lac)169, ptr3, degP41, ompT, kan^(r)) (U.S. Pat. No.5,288,931, ATCC No. 55,244), Bacillus subtilis, Salmonella typhimurium,Serratia marcesans, and Pseudomonas species.

In addition to prokaryotes, eukaryotic organisms, such as yeasts, orcells derived from multicellular organisms can be used as host cells.For expression in yeast host cells, such as common baker's yeast orSaccharomyces cerevisiae, suitable vectors include episomallyreplicating vectors based on the 2-micron plasmid, integration vectors,and yeast artificial chromosome (YAC) vectors. For expression in insecthost cells, such as Sf9 cells, suitable vectors include baculoviralvectors. For expression in plant host cells, particularly dicotyledonousplant hosts, such as tobacco, suitable expression vectors includevectors derived from the Ti plasmid of Agrobacterium tumefaciens.

However, interest has been greatest in vertebrate host cells. Examplesof useful mammalian host cells include monkey kidney CV1 linetransformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line(293 or 293 cells subcloned for growth in suspension culture, Graham etal., J. Gen Virol., 36: 59 (1977)); baby hamster kidney cells (BHK, ATCCCCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub and Chasin,Proc. Natl. Acad. Sci. USA, 77: 4216 (1980)); mouse sertoli cells (TM4,Mather, Biol. Reprod., 23: 243-251 (1980)); monkey kidney cells (CV1ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCCCRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); caninekidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCCCRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (HepG2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells(Mather et al., Annals N.Y. Acad. Sci., 383: 44-68 (1982)); MRC 5 cells;FS4 cells; and a human hepatoma cell line (Hep G2). For expression inmammalian host cells, useful vectors include vectors derived from SV40,vectors derived from cytomegalovirus such as the pRK vectors, includingpRK5 and pRK7 (Suva et al., Science, 237: 893-896 (1987), EP 307,247(Aug. 15, 1989), EP 278,776 (Aug. 17, 1988)) vectors derived fromvaccinia viruses or other pox viruses, and retroviral vectors such asvectors derived from Moloney's murine leukemia virus (MoMLV).

Optionally, the DNA encoding the Z domain variant of interest isoperably linked to a secretory leader sequence resulting in secretion ofthe expression product by the host cell into the culture medium.Examples of secretory leader sequences include stII, ecotin, lamB,herpes GD, 1pp, alkaline phsophatase, invertase, and alpha factor. Alsosuitable for use herein is the 36 amino acid leader sequence of proteinA (Abrahmsen et al., EMBO J., 4: 3901 (1985)).

Host cells are transfected and preferably transformed with theabove-described expression or cloning vectors of this invention andcultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences.

Transfection refers to the taking up of an expression vector by a hostcell whether or not any coding sequences are in fact expressed. Numerousmethods of transfection are known to the ordinarily skilled artisan, forexample, CaPO₄ precipitation and electroporation. Successfultransfection is generally recognized when any indication of theoperation of this vector occurs within the host cell.

Transformation means introducing DNA into an organism so that the DNA isreplicable, either as an extrachromosomal element or by chromosomalintegrant. Depending on the host cell used, transformation is done usingstandard techniques appropriate to such cells. The calcium treatmentemploying calcium chloride, as described in section 1.82 of Sambrook etal., Molecular Cloning (2nd ed.), Cold Spring Harbor Laboratory, NY(1989), is generally used for prokaryotes or other cells that containsubstantial cell-wall barriers. Infection with Agrobacterium tumefaciensis used for transformation of certain plant cells, as described by Shawet al., Gene, 23: 315 (1983) and WO 89/05859 published Jun. 29, 1989.For mammalian cells without such cell walls, the calcium phosphateprecipitation method described in sections 16.30-16.37 of Sambrook etal., supra, is preferred. General aspects of mammalian cell host systemtransformations have been described by Axel in U.S. Pat. No. 4,399,216issued Aug. 16, 1983. Transformations into yeast are typically carriedout according to the method of Van Solingen et al., J. Bact., 130: 946(1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76: 3829 (1979).However, other methods for introducing DNA into cells such as by nuclearinjection, electroporation, or by protoplast fusion may also be used.

Prokaryotic host cells used to produce the present Z domain variantpeptides can be cultured as described generally in Sambrook et al.,supra.

The mammalian host cells used to produce the Z domain variant peptidesof the invention can be cultured in a variety of media. Commerciallyavailable media such as Ham's F10 (Sigma), Minimal Essential Medium((MEM), Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle'sMedium ((DMEM), Sigma) are suitable for culturing the host cells. Inaddition, any of the media described in Ham and Wallace, Meth. Enz., 58:44 (1979), Barnes and Sato, Anal. Biochem., 102: 255 (1980), U.S. Pat.Nos. 4,767,704; 4,657,866; 4,927,762; or 4,560,655; WO 90/03430; WO87/00195; U.S. Pat. Re. 30,985; or U.S. Pat. No. 5,122,469, thedisclosures of all of which are incorporated herein by reference, may beused as culture media for the host cells. Any of these media may besupplemented as necessary with hormones and/or other growth factors(such as insulin, transferrin, or epidermal growth factor), salts (suchas sodium chloride, calcium, magnesium, and phosphate), buffers (such asHEPES), nucleosides (such as adenosine and thymidine), antibiotics (suchas Gentamycin™ drug), trace elements (defined as inorganic compoundsusually present at final concentrations in the micromolar range), andglucose or an equivalent energy source. Any other necessary supplementsmay also be included at appropriate concentrations that would be knownto those skilled in the art. The culture conditions, such astemperature, pH, and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan.

The host cells referred to in this disclosure encompass cells in invitro culture as well as cells that are within a host animal.

In an intracellular expression system or periplasmic space secretionsystem, the recombinantly expressed Z domain variant peptide can berecovered from the culture cells by disprupting the host cellmembrane/cell wall (e.g. by osmotic shock or solubilizing the host cellmembrane in detergent). Alternatively, in an extracellular secretionsystem, the recombinant peptide can be recovered from the culturemedium. As a first step, the culture medium or lysate is centrifuged toremove any particulate cell debris. The membrane and soluble proteinfractions are then separated. The Z domain variant peptide can then bepurified from the soluble protein fraction. If the peptide is expressedas a membrane bound species, the membrane bound peptide can be recoveredfrom the membrane fraction by solubilization with detergents. The crudepeptide extract can then be further purified by suitable procedures suchas fractionation on immunoaffinity or ion-exchange columns; ethanolprecipitation; reverse phase HPLC; chromatography on silica or on acation exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammoniumsulfate precipitation; gel filtration using, for example, Sephadex G-75;hydrophobic affinity resins and ligand affinity using IgG ligandimmobilized on a matrix.

In cyclized embodiments of the invention, the recombinantly produced Zdomain variant peptide can be cyclized by formation of an intramoleculardisulfide bond as described in Section B(II)(1)(b) above.

III. Z Domain Variant Peptide Ligands for Affinity Chromatography of IgG

In one embodiment, the Z domain variant peptides are used as ligands inthe affinity chromatography of IgG, IgG fusion proteins(immunoadhesins), and IgG-bearing cells. The Z domain variant peptidescan be made as described in Section B(II) above. Next, the selectedpeptide can be coupled to a suitable macromolecular solid phase matrix,such as cellulose, cross-linked dextran derivatives (e.g. Sepharose),polyacrylamides, porous glass and ceramics, by any convenient method.

A commonly employed technique for attaching peptide ligands topolysaccharide matrices, e.g. agarose, dextran or cellulose, involvesactivation of the carrier with cyanogen halides and subsequent couplingof the peptide ligand's primary alphatic or aromatic amines to theactivated matrix. The activation of polysaccharides with cyanogenbromide (CNBr) at alkaline pH was introduced to affinity chromatographyby Axen et al., Nature, 214: 1302 (1967). In one aspect of theinvention, the activation of polysaccharide matrices, particularlyagarose matrices, is performed according to the titration-activationmethod. In this procedure, for example, 20 g of exhaustively washedmoist agarose cake is added to 20 ml of water in a 100 ml beakerequipped with a 0-100° C. thermometer, a pH meter and a 25 mm magneticstirring bar. The suspension is stirred slowly, the temperature loweredto about 10-15° C. by the addition of crushed ice and the pH adjusted to10.8±0.1 by the addition of 1-2 drops of 4 N NaOH. The activationprocedure is initiated by the addition of the CNBr and the pH of thereaction maintained at 10.8±0.1 by manual titration with the 4 N NaOH.The CNBr (100 g/mg moist weight gel) can be added as a crystallinesolid, a crushed solid, an aqueous solution or by adding an aliquot of astock solution. The latter can be prepared by dissolving CNBr inacetonitrile (1 g/ml) and storing in a tightly stoppered vial at −20° C.The temperature is subsequently allowed to rise to 18-20° C.

Despite the relative simplicity of the titration method, it may bepreferable to use the faster and technically simplified method of Marchet al., Anal. Biochem., 60: 149 (1974). The activation procedure isperformed in concentrated carbonate buffer. The required amount ofwashed gel is suspended in an equal volume of 2 M NaHCO₃-NaCO₃ buffer(pH 10.9) in a beaker equipped with a thermometer and magnetic stirringbar. The slurry is cooled to approximately 4-5° C., the activated gel istransferred to a sintered funnel and washed.

The concentration of CNBr recommended in the procedures described aboveis satisfactory for moderate levels of ligand substitution. When loweror higher levels of activation are required, 50 mg and 200-300 mg CNBr/gmoist weight gel respectively can be employed together with 2 M and 8 MNaOH for the titration.

It is generally recognized that the CNBr-activated intermediatefunctional groups of polysaccharide gels display limited stability andtherefore it is preferable that the gel be washed as rapidly as possibleprior to transferring the gel to the coupling-reaction medium. At theend of the activation step, the gel is rapidly cooled by the addition ofcrushed ice and poured into a large sintered glass funnel which has beenpre-cooled with crushed ice. The suspension is rapidly filtered into aBuchner flask (2 liter) containing solid ferrous sulfate to removeunreacted CNBr and cyanides as harmless ferrocyanide. The gel issubsequently washed under suction with 1 liter ice-cold distilled waterand 1 liter of the buffer to be used in the coupling stage, typicallyice-cold 0.1 M NaHCO₃-NaCO₃ buffer (pH 8.5-9.5).

CNBr-activated Sepharose 4B is available commercially from Pharmacia andobviates the hazardous manipulation of CNBr. The activated gel is freezedried in the presence of dextran and lactose to preserve the beaded formand supplied in 15 g air-tight packs. The required amount offreeze-dried powder is swollen in 1 mM HCl on a glass filter and washedwith at least 200 ml of the same solution per gram of powder. 1 g offreeze-dried material is roughly equivalent to 3.5 ml final gel volume.The ligand-binding capacity of the gel is conserved more effectively bywashing with solutions of low pH than with solutions of pH>7. The gel isthen ready to couple ligand as soon as the washing is completed.

Pharmacia also markets CNBr-activated Sepharose 6 MB for use in cellbiology and immunology for the separation of “functionally homogeneouscell populations”. It is produced by activation of Sepharose 6MBmacrobeads (diameter 200-300 μm) with cyanogen bromide and is handled ina manner analogous to CNBr-activated Sepharose 4B.

The peptide to be coupled is suspended in a volume of the cold bufferequal to the volume of the packed gel, added to the moist, washed gel,and then the suspension is immediately mixed (in a Buchner funnel) witha glass stirring rod. The entire procedure of washing, adding thepeptide solution, and mixing preferably consumes less than 90 seconds.The suspension is transferred from the Buchner funnel to a beakercontaining a magnetic mixing bar and is gently stirred at 4° C. Althoughthe reaction is essentially complete in 2 to 3 hours, the mixture isallowed to stand at 4° C. for 16 to 20 hours to insure complete loss ofreactive polysaccharide groups. The peptide-linked gel is then washedwith large volumes of water until it is established that peptide is nolonger being removed.

The quantity of peptide ligand coupled to the polysaccharide gel can inpart be controlled by the amount of peptide added to the activatedmatrix. When highly substituted polysaccharide gel derivatives aredesired, the amount of peptide added should be 20 to 30 times greaterthan that which is desired in the final product. For ordinaryprocedures, 100 to 150 mg of cyanogen bromide are used per ml of packedpolysaccharide gel, but much higher coupling yields can be obtained ifthis amount is increased to 250 to 300 mg. The pH at which the couplingreaction is performed also affects the degree of coupling, since it isonly the unprotonated form of a peptide's amino groups that reacts withCNBr-activated polysaccharides. Preferably, the N-terminal α-amino groupof the peptide ligand is used for coupling with the activatedpolysaccharide matrix. α-amino groups will couple optimally at a pH ofabout 9.5 to 10.0. If coupling at the ε-amino group(s) of the selectedpeptide ligand (such as the ε-amino groups of the lysinyl residues) isdesired, the coupling reaction should be conducted at a pH value ofabout 10.0, and a large excess of peptide should be added. If couplingat the aromatic amino groups in the histidyl or tryptophanyl residues ofthe selected peptide is desired, very high coupling efficiency can beobtained at pH values between 8 and 9.

In another embodiment, a spacer molecule is interposed between thepeptide ligand and the matrix backbone. For example, it is possible touse the bromoacetyl, diazonium, or sulfhydryl polysaccharide matrixderivatives described in Cuatrecasas, J. Biol. Chem., 245: 3059-3065(1970) to couple the selected peptide to a solid support by means of anarm extending some distance from the matrix backbone. Alternatively, thespacer arms described in Lowe, “An Introduction to AffinityChromatography”, in Laboratory Techniques in Biochemistry and MolecularBiology, Work and Work, eds, North-Holland Publishing Co. (New York:1979), pp.344-400 can be used to link the peptides of the invention topolysaccharide matrices.

The matrix coupled to the Z domain variant peptides of the invention canbe used for affinity chromatographic purification of IgG, IgG fusionproteins or IgG-bearing cells by any of the techniques well known andwidely used for protein A affinity chromatography. Since the peptides ofthe invention possess IgG-binding activities similar to that of wildtype protein A, affinity chromatography with the insolubilized peptideligand can be performed under the same or similar conditions as affinitychromatography with insolubilized protein A.

In one embodiment for affinity purification of IgG or IgG fusionproteins, a gravity-feed operated column of the desired size is packedwith the affinity gel and washed with at least 5 bed volumes ofTris-saline Tween 20 (50 mM Tris buffer, pH 7.6, 150 mM NaCl and 0.05%Tween 20) (TST) prior to use. The column can then be equilibrated in 2-3bed volumes each of 1) 0.5 M CH₃OOH (HAc) adjusted to pH 3.4 withNH₄CH₃COOH (NH₄Ac); 2) TST; 3)0.5 M HAc, pH 3.4; and 4) TST. The pH ofthe column eluate is determined, and the pH of the sample, cellsupernatant or clarified growth medium is adjusted to match, ifnecessary. A suitable aliquot of the sample is applied to the top of amoist bed of the affinity adsorbent, allowed to run in, and the columnis washed with 1) 10 bed volumes of TST and 2) 2 bed volumes of 5 mMNH₄Ac, pH 5.0. Next, the sample can be eluted from the column with 0.5 MHAc, pH 3.4 and aliquots of eluate collected and peak fractionsidentified by spectrophotometry at 280 nm. Alternatively, samples can beeluted from the column with concentrated solutions of the same peptideused as the column adsorbent.

It will be appreciated that affinity purification techniques using the Zdomain variant peptides of the invention are not limited to columnchromatographic procedures. In many cases, it may be preferable to use abatchwise technique. When relatively small amounts of IgG or IgG fusionprotein are to be extracted from a mixture containing a significantproportion of inert protein, the purification may be achieved morereadily by adding a slurry of the adsorbent. The non-adsorbed proteinscan be washed off either under batchwise conditions or by placing theadsorbent in a chromatographic column and proceeding as described above.

IV. Z Domain Variant Peptide Affinity Handles for IRG Ligand AffinityChromatograph of Fusion Proteins

The ability of the Z domain variant peptides of the invention to bind tothe Fc portion of most mammlian class G immunoglobulins makes themuseful as “affinity handles” in gene fusion expression systems. Thisstrong and specific affinity permits the purification of the fusionproteins in a single step by IgG affinity chromatography. The Z domainvariant peptide “handle” can later be removed to release the desired,native sequence protein product. It has also been shown that manyheterologous proteins are degraded when expressed directly in E. coli,but are stable when expressed as fusion proteins (Marston, Biochem. J.,240: 1 (1986)).

The question of whether to remove the peptide purification handle (tail)is essentially dependent on the end-use of the selected or desiredprotein. Accordingly, the invention encompasses fusion proteins thateither are or are not designed to facilitate the removal of the Z domainvariant peptide to which the desired protein is fused. For lab-scalecharacterization of a protein that has previously been difficult toobtain in sufficient quantities, it can be convenient to leave thepeptide “tail” on, after initial demonstration that the tail does notinterfere with the biological function of the desired protein. Ifinterference is encountered, removal of the tail may be necessary inorder to obtain reliable results. In some applications, a slight or evenconsiderable loss of activity can be justified by the ease ofpurification and assayability provided by the fusion moiety. Selectedproteins in this category may include industrial enzymes, diagnosticproteins, and enzymes that are to be immobilized. In the latter case thetail could be designed to promote simultaneous purification andimmobilization by binding to a solid support. For pharmaceuticalapplications, precise removal of the fusion tail is usually desired inorder to achieve absolute product authenticity.

When a native gene product is desired, a site-specific cleavage of thefusion protein must be performed. There are two principal ways to obtainspecific cleavages of proteins: chemical and enzymatic. Examples ofchemical cleavage agents include hydroxylamine, formic acid, aceticacid, cyanogen bromide, NBPS-skatole, o-iodosobenzoic acid, andN-chlorosuccinimide. Chemical agents can be used in the site-specificcleavage of fusion proteins as described in Carter, “Site-SpecificProteolysis of Fusion Proteins”, in Protein Purification: From MolecularMechanisms to Large Scale Processes, Ladisch et al., eds, AmericanChemical Society Symposium Series No. 427, Ch. 13, pp. 181-193 (1990).Alternatively, fusion tails can be removed by cleavage with enzymes,such as chymotrypsin, collagenase, endoprotease Lys-C, enterokinase,factor Xa, kallikrein, renin, H64A subtilisin BPN′, thrombin, trypsinand ubiquitin protein peptidase as described in Carter, supra.

Enzymatic cleavages are more affected by steric factors than chemicalmethods. Accordingly, it is preferred that the cleavage site becarefully engineered to be structurally accessible to the enzyme. Onestrategy for enhancing the accessiblity to proteases is to flank thetarget sequence on both sides with short stretches of glycine residuesor on one side if the correct terminus of the protein of interest isrequired after cleavage. An alternative strategy to enhance substrateaccessibility is to perform the digests under denaturing or reducingconditions (provided that this is compatible with the protease used) orby denaturation of the substrate prior to digestion.

The invention encompasses fusion proteins in which the present Z domainvariant peptides are fused to either the C-terminus or the N-terminus,or both terminii, of the desired protein. If removal of some or all ofthe affinity handle peptide sequence is desired, a protease-specificcleavage site can be engineered into the fusion protein at or near thefusion joint between the affinity handle peptide and the desiredprotein. In the case of a fusion protein with the affinity handle fusedto the N-terminus of the desired protein (N-terminal fusion proteins),the affinity handle can advantageously comprise a Z domain variantpeptide wherein the peptide either comprises a specific cleavage site oris attached at its C-terminus to a specific cleavage site such that theaffinity handle sequence can be substantially removed from the desiredprotein by site-specific proteolytic cleavage. The cleavage site can berecognizable by either a chemical cleavage agent or an enzymaticcleavage agent, and is preferably not found in the amino acid sequenceof the desired protein, i.e. cleavage with the agent does not result infragmentation of the desired protein product. Enzymatic cleavage is wellsuited for production of an authentic N-terminus for the desired proteinin the case of N-terminal fusion proteins because the major specificitydeterminants for enzyme endoproteases are associated with residues onthe N-terminal side of the scissile bond, not the C-terminal side of thescissile bond. The non-specificity on the C-terminal side of thescissile bond permits the design of N-terminal fusion proteins in whichthe specific cleavage site directly abuts the N-terminal residue of thedesired protein.

In the case of a fusion protein with the affinity handle fused to theC-terminus of the desired protein, the affinity handle canadvantageously comprise a Z domain variant peptide wherein the peptideeither comprises a specific cleavage site or is attached at itsN-terminus to a specific cleavage site such that the affinity handlesequence can be substantially removed from the desired protein bysite-specific proteolytic cleavage. The cleavage site can berecognizable by either a chemical cleavage agent or an enzymaticcleavage agent, and is preferably not found in the amino acid sequenceof the desired protein, i.e. cleavage with the agent does not result infragmentation of the desired protein product.

Fusion proteins containing the Z domain variant peptides of theinvention can be conveniently produced by any of the methods describedin Section B(II) above. Preferably, the fusion protein of interest isproduced by synthesizing DNA encoding the fusion protein, constructingan expression vector in which the coding sequence is operably linked tocontrol sequences recognized by a host cell, transforming the host cellwith the recombinant expression vector, culturing the transformed hostcell under conditions wherein the fusion protein is expressed, andrecovering the expressed fusion protein as described in Section B(II)above. In the case of a host cell secretion system, the cell culturefluid can be harvested, filtered, loaded onto an IgG-ligand affinitychromatography column, and the bound fusion protein can be eluted fromthe column and collected using the same procedures as those employed foraffinity purification of IgG with the Z domain variant-ligand affinitycolumns described in Section B(III) above. In one embodiment, an IgGSepharose 6 Fast Flow (Pharmacia, Sweden) affinity column is used topurify the fusion protein according to the manufacturer's instructions.In the case of an intracellular expression system or a periplasmic spacesecretion system, the host cells are disrupted or lysed, the cell debrisis preferably removed from the lysate, e.g. by centrifugation, and thecrude extract is passed over an IgG affinity column and the fusionprotein recovered as described above.

The desired protein may or may not be be properly folded when expressedas a fusion protein. Also, in embodiments employing a specific cleavagesite allowing removal of the affinity handle from the desired protein,the specific cleavage site may or may not be accessible to the proteaseused for cleavage. These factors determine whether the fusion proteinmust be denatured and then refolded, and if so, whether these proceduresare employed before or after cleavage. When denaturation and refoldingare needed, typically the protein is treated with a chaotrope, such as aguanidine hydrochloride, and is then treated with a redox buffer,containing, for example, reduced and oxidized dithiothreitol orglutathione at the appropriate ratios, pH, and temperature, such thatthe protein of interest is refolded to its native conformation.

Likewise, in embodiments of the invention employing cyclized Z domainvariant peptides as affinity handles for fusion proteins, thecyclization of the affinity handle peptide may require denaturationfollowed by gradual renaturation of the fusion protein recovered in theevent that misfolding or inappropriate disulfide bond formation hasoccurred in the fusion protein product. As above, when denaturationand/or disulfide bond exchange is needed, the fusion protein istypically treated with a chaotrope, such as a guanidine hydrochloride,and is then treated with a redox buffer, containing, for example,reduced and oxidized dithiothreitol or glutathione at the appropriateratios, pH, and temperature, such that the protein of interest isrefolded to its native conformation and the desired cyclization of theaffinity handle peptide occurs.

V. Therapeutic Uses of Z Domain Variant Peptides

Protein A most likely contributes to the pathogenicity of Staphylococcusaureus by its IgG binding activity (Patel et al., Infect. Immun., 55:3103 (1987)). Protein A molecules on the surface of staphylococcalpathogens can cause the host's IgG to coat the bacterium such that theeffector functions of the immune system are incapable of recognizingforeign antigens on the bacterial cell surface and are incapable ofinteracting with the Fc regions of the IgG molecules coating thebacterium. Thus, the IgG-coated staphylococcus would be “invisible” tothe host's immune defenses. The Z domain variant peptides of theinvention can be used to strip IgG from the surface of staphylococcalpathogens by out-competing the staphylococcal cell surface-bound proteinA molecules for interaction with IgG. In this way, the Z domain variantpeptides would unmask the foreign antigens on the bacterial cell surfacefor recognition by the host's immune system.

Accordingly, the invention provides for the treatment or prophylaxis ofdiseases mediated by staphylococcal pathogens, including staphylococcalfurunculosis, impetigo, pyemia, osteomyelitis, suppuration of wounds,food poisoning, staphylococcemia, and any other staphylococcicinfection, by administering an effective amount of the compounds of theinvention to a patient in need of such treatment or prophylaxis. Alsoprovided herein are compositions containing an effective amount of thecompounds of the invention, including the nontoxic addition salts,amides and esters thereof, which may, alone, serve to provide theabove-recited therapteutic or prophylactic benefits. Such compositionscan be provided together with physiologically tolerable liquid, gel orsolid diluents, adjuvants and excipients.

In addition, the Z domain variant peptides of the invention can also beused for the treatment or prophylaxis of other disease states that areinduced or mediated by the Fc region of IgG (e.g. conditions such asrheumatoid arthritis).

The compounds and compositions can be adminstered to humans in a mannersimilar to other therapeutic agents. The dosage to be administered willdepend on the usual factors, including the age, weight, sex, andcondition of the patient and the route of administration,. In general,the dosage required for therapeutic efficacy will range from about 0.01to 1000 μg/kg, more usually 0.1 to 25 μg/kg of the patient's bodyweight. Alternatively, dosages within these ranges can be administeredby constant infusion over an extended period of time until the desiredtherapeutic benefits have been obtained.

Typically, such pharmaceutical compositions are prepared as injectableliquid solutions or suspensions. Compositions may also be emulsified.The active ingredient is often mixed with diluents or excipients whichare physiologically tolerable and compatible with the active ingredient.Suitable diluents and excipients are, for example, water, saline,dextrose, glycerol, or the like, and combinations thereof. In addition,if desired the compositions may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, stabilizing orpH-buffering agents, and the like. For a more detailed description ofthe foregoing see a standard pharmaceutical text such as Remington'sPharmaceutical Sciences, Mack Publishing Co. Easton, Pa. (1970).

The pharmaceutical compositions of this invention are conventionallyadministered parenterally by injection, either subcutaneously orintravenously. Additional formulations which are suitable for othermodes of administration include suppositories, intranasal aerosols, and,in some cases, oral formulations. For suppositories, traditional bindersand excipients may include, for example, polyalkylene glycols ortriglycerides; such suppositories may be formed from mixtures containingthe active ingredient in the range of 0.5% to 10% preferably 1%-2%. Oralformulations include such normally employed excipients as, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharin, cellulose, magnesium carbonate, and the like. Thesecompositions take the form of solutions, suspensions, tablets, pills,capsules, sustained-release formulations, or powders, and contain10%-95% if active ingredient, preferably 25%-70%.

The peptide compounds of the invention may be formulated into thecompositions as neutral or salt forms. Pharmaceutically acceptablenontoxic salts include the acid salts (formed with the free aminogroups) and which are formed with inorganic acids such as, for example,hydrochloric or phosphoric acids, or organic acids such as acetic,oxalic, tartaric, mandelic, and the like. Salts formed with the freecarboxyl groups may be derived from inorganic bases such as, forexample, sodium, potassium, ammonium, calcium, or ferric hydroxides, andsuch organic bases as isopropyl amine, 2-ethylamino ethanol, histidine,procaine, and the like.

Therapy of staphylococcal diseases with the compositions of theinvention can be combined with other known therapies forstaphylococcias, such as treatment with penicillins, cephalosporins,polymyxins, nitroaromatics, aminoglycosides, tetracyclines, macrolides,lincomycins, and the like, or any other agents traditionally used forsuch infections.

Further details of the invention can be found in the following examples,which further define the scope of the invention. All references citedthroughout the specification, and the references cited therein, arehereby expressly incorporated by reference in their entirety.

EXAMPLE 1

The B-domain of protein A, and a more stable variant called the Z-domain(Nilsson, B. et al., Protein Engng. 1: 107-113 (1987)), are three-helix,59 residue modules that bind the F_(c)-portion of IgG's with a K_(d) ofabout 10-50 nM (Cedergren, L. et al., Protein Engng. 6: 441-448 (1993))X-ray (Deisenhofer, J., Biochemistry 20: 2361-2370 (1981)), NMR (Gouda,H. et al., Biochemistry 31: 9665-9672 (1992)) and mutational studies(Cedergren, L. et al., supra) show that binding contacts are presentedfrom helix-1 (residues 7-18) and helix-2 (residues 20-38) (FIG. 1).Nonetheless, when helix-3is deleted the remaining peptide loses itsα-helical character and binding affinity is reduced >10⁵-fold (Huston,J. S., et al., Biophys. J., 62: 87-91 (1992)). It was determined hereinthat the two-helix motif can be stabilized and affinity optimized byimproving three regions of the 38-residue peptide: the exposedhydrophobic region from helix-1 and helix-2 that contacts helix-3 (theExoface, FIG. 2A), the buried residues between helix-1 and helix-2 (theIntraface, FIG. 2B), and the residues from the Z-domain predicted tocontact the IgG (the Interface, FIG. 2C).

Phage display of protein or peptide libraries offers a powerfulmethodology for the selection of novel binding partners with improvedaffinity, altered specificity, or improved stability (Smith, G. P.,Curr. Opin. Biotechnol. 2: 668-673 (1991)). High affinity proteins,displayed in a monovalent fashion as fusions with the M13 gene III coatprotein (Clackson, T., et al., Trends Biotechnol. 12: 173-183 (1994)),can be identified by cloning and sequencing the corresponding DNApackaged in the phagemid particles after a number of rounds of bindingselection.

A functional Z-domain of Protein A can be displayed on M13 phageparticles (Nord, K., et al., Protein Engng. 8,: 609-614 (1995) andDjojonegoro, B. M. et al., Biol/Technology. 12: 169-172 (1994)) and thisbinds with an EC₅₀ of about 20 nM to IgG. Starting with a peptiderepresenting the first two helices of the Z-domain, phagemid librarieswere constructed to allow selection of variants at the Exoface,Intraface, and Interface regions. The libraries were selectedsequentially and combined such that the best selectant from the Exofacelibrary was used as a starting scaffold for the Intraface library, andthe best selectant from that was incorporated into the starting scaffoldfor the Interface library. Through successive compilation of selectedmutations, a peptide was engineered to bind to IgG₁ with nearly the sameaffinity as the wild type Z-domain at about half the size.

Materials and Methods

Construction of Libraries: Monovalent phagemid libraries (Lowman, H. B.et al., Methods: Companion Methods Enzymol. 3: 205-216 (1991)) of thetruncated Z peptide were generated by site directed mutagenesis (Kunkel,T. A., et al., Methods Enzmol. 204: 125-139 (1991)). Each librarycontained four or five codons fully randomized for all 20 amino acids(as shown in Table 1 below). The starting template for libraries 2 andincluded a frame shift as well as a TAA stop codon to eliminate thebackground wild type clones. Stocks of approximately 10¹⁴ phagemid perML were prepared from PEG precipitates of culture broths from XL-1 Bluecells containing the plasmid and superinfected with KO7 helper phage.

TABLE 1 Consensus residues from each truncated Z-domain library^(a). WTresidue Selected residues P_(e) P_(f) (P_(f)-P_(e))/σ Exoface-1Library^(b) I17 I 0.031 0.47 10.5 A 0.062 0.53 8.1 L23 D 0.031 0.67 15.6N 0.031 0.17 3.4 L23 L 0.094 0.94 12.3 F31 K 0.031 0.47 10.5 F 0.0310.18 3.5 Intraface-2 Library^(c) A13 R 0.094 0.85 11.6 I17 A 0.062 1.017.4 R28 R 0.094 1.0 13.9 I32 I 0.031 1.0 24.8 L35 I 0.031 0.90 22.3Interface-3A Library^(d) D3 A 0.062 0.40 4.4 R 0.094 0.40 3.3 N4 N 0.0310.50 8.7 Q 0.031 0.30 5.0 K5 G 0.062 0.60 7.1 S 0.094 0.30 2.2 F6 F0.031 1.0 17.9 Interface-3B Library F6 F 0.031 0.60 10.5 G 0.062 0.404.4 N7 N 0.031 0.40 6.8 W 0.031 0.40 6.8 K8 M 0.031 0.90 16.1 E9 Q 0.0310.40 6.8 K 0.031 0.20 3.1 Interface-3C Library Q10 Q 0.031 1.0 17.9 Q11Q 0.031 1.0 17.9 N12 R 0.094 0.70 6.6 E 0.031 0.20 3.1 R13 R 0.094 0.605.5 A 0.062 0.30 3.1 Interface-3D Library F14 F 0.031 1.0 17.9 Y15 Y0.031 1.0 17.9 L18 L 0.094 1.0 9.8 H19 H 0.031 1.0 17.9 Interface-3ELibrary N29 N 0.031 1.0 17.9 A30 A 0.062 1.0 12.3 Q33 K 0.031 0.80 14.2K36 R 0.094 1.0 9.8 ^(a)The sequence of the original 38 residue peptidederived from the Z-domain was: AVDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDD(SEQ ID NO: 47). A single oligonucleotide was used to generate randommutations except for the Intraface-2 library which introduced twooligonucleotides simultaneously. Randomized codons were synthesized asNNS, where N represents any of the four bases and S represents an equalmix of G/C. This generates 32 possible codons encoding all 20 aminoacids and theoretically produces #32^(n) possible DNA sequences. Thenumber of transformants in each library greatly exceeded the theoreticalvalue in all cases except the Intraface-2 library where 3.4 × 10⁷possible sequences exist but only 2 × 10⁶ transformants were obtained.Phagemid libraries were constructed and sorted for binding toimmobilized IgG according to the Materials and Methods and Table 1.After various rounds of binding selection, clones were sequenced andscored for the number of times the #most commonly selected residuesappeared at each of the mutated positions. P_(e) = number of possibleNNS codons/32 (for example, Ile is 1/32 and Arg is 3/32, etc.); P_(f) =number of times residue found/number of clones sequenced; standarddeviations σ_(n) = [P_(e)(1-P_(e))/n]^(1/2); n = number of clonessequenced; residues for which [P_(f)-P_(e))/σ_(n)] < 2.0 are not shown.^(b)The Exoface-1 library was carried through four rounds of selectionand eighteen clones were sequenced. Seven of the L23 codons found werenon NNS codons, and thus were derived from the wild-type template; oneclone was illegible at two positions. ^(c)The Intraface-2 library wascarried through ten rounds of selection and twenty clones weresequenced. Five clones were sequenced at round three, and ten at roundsix to monitor library diversity. ^(d)The Interface-3A through 3Elibraries were carried through ten rounds of selection and ten clonesfrom each were sequenced. Five clones were sequenced from each libraryat round three, and ten clones from each at round six to monitor librarydiversity.

Selection and analysis of IgG specific phagemids: Microtiter plates(Nunc Maxisorb, 96 well) were coated with human IgG (Zymed) at aconcentration of 10 μg per mL in 5 mM sodium carbonate pH 9.6 overnightat 4° C. Wells were blocked with a 1:1 mixture of 50 mM sodium carbonate(pH 9.6) and binding buffer (phosphate buffered saline (pH 7.2) PBS,with 0.1% BSA (Sigma, globulin free) and 0.05% Tween 20 (Sigma) for 1hour. Approximately 10¹² phage from the appropriate stock diluted to 100μL with binding buffer were incubated for 2 hours before washing 20times with PBS containing 0.05% Tween 20. Bound phage were eluted with100 μL of 0.2 M glycine pH 2.0, neutralized with 1M Tris pH 9.0 and thenused to infect E. coli (XL-1 Blue, Stratagene) for phagemid production.Phage ELISA were determined according to the method of Cunningham, B. C.et al., EMBO J., 13: 2508-2515 (1994) against human IgG coated at 10 μgper mL in microtiter plates using an anti-M13-horseradish peroxidaseconjugate (Pharmacia) with an o-phenylene diamine substrate (Sigma).Clones of interest were transformed into 27C7 cells (a non-suppressorstrain of E. coli) (U.S. Pat. No. 5,288,931; ATCC No. 55,244) and 250 mLcultures were grown in low phosphate AP5 minimal media for 16 hoursaccording to method of Chang, C. N., et al., Gene 55: 189-196 (1987).Both the supernatants and the periplasmic shockates were purified byaffinity chromatography on IgG-Sepharose (Pharmacia). Final purificationwas accomplished by reverse phase HPLC. The mass of each peptide wasconfirmed by electrospray mass spectrometry and the peptides weredetermined to be >95% pure by HPLC. Peptide concentrations weredetermined by quantitative amino acid analysis.

Binding Kinetics and Circular Dichroism studies: Association anddissociation rate constants for the binding of both Z-domain and theselected peptides were determined by surface plasmon resonance. Amonoclonal IgG₁ was immobilized on the biosensor chip covalently throughthe primary amines according to the method of Johnsson, B. E et al.,Anal. Biochem. 198: 268-277 (1991). A coupling density of approximately6000 RUs was used for both association and dissociation constantdeterminations. Association and dissociation rates shown in Table 2below were measured at flow rates of 20 and 25 μL per minuterespectively in PBS buffer (pH 7.4) with 0.05% Tween 20 as described byKarlsson, R. et al., J. Immunol. Methods. 145: 229-240 (1991).

CD spectra were recorded on an AVIV 60DS spectropolarimeter in thewavelength range of 250-190 nm in 0.2 nm intervals in a thermostatedcircular cuvette with a path length of 0.05 cm. The final CD spectrarepresent an average of 3 scans with an integration time of 2 seconds.Results are reported as mean residue ellipticity (^(Θ)MRW, indeg-cm²-dmol⁻¹). Spectra were recorded at 8° C. with peptideconcentrations of 0.20 mg/mL in 100 mM sodium chloride, 10 mM Tris-HClat pH 7.2. Curve fitting was accomplished using the method of Provencherand Glockner in Biochemistry, 20: 33-37 (1981).

Results and Discussion

The Exoface Selectants: For the Exoface library, four residues fromhelix-1 and helix-2 (Ile17, Leu20, Leu23 and Phe31) that form ahydrophobic core with helix-3 in the intact Z-domain (FIG. 2A) weremutated. After four rounds of binding selection to IgG, a clearconsensus was seen as shown in Table 1 above. The wild-type residues,Leu20 and Phe31, were replaced by the charged residues Asp and Lys,respectively (FIG. 2A). At the other two positions the wild-typeresidues, Ile17 and Leu23, were predominantly retained, possibly becausethey stabilize the hydrophobic intraface between helix-1 and helix-2 orthe type 1 β-turn that connects them. The consensus Exoface-1 selectant(L20D/F31K) exhibited an EC₅₀ by phage ELISA of 3.4 μM for binding toIgG as shown in Table 2 below. The truncated Z-domain peptide (residues1-38) did not show any detectable binding by phage ELISA although aK_(d) in the millimolar range has been reported for an analogous peptide(Huston, J. S., et al., supra).

TABLE 2 Phage ELISA and binding kinetics for representative selectantsand consensus peptides. k_(on) k_(off) K_(d) EC₅₀ Protein (×10⁵M⁻¹ s⁻¹)(s⁻¹) (nM) (nM) 3-helix Z domain 2.09 0.0021  10  20 2-helix Z-domain NDND >1 × 10⁴ >1 × 10⁴ Exoface-1 variant: A = L20D/F31K ND ND ND 3400 Exoface-1 plus Intraface-2 variants: B = A + I17A/L35A ND ND ND 930 C =A + A13R/I17A/ 1.78 0.133 750 420 L35I Exoface-1 plus Intraface-2 plusInterface-3 variants: D = C + D3R/K5G 2.06 0.091 440 230 E = C +D3A/N4Q/ 1.61 0.091 570 140 K5S F = C + K8M/E9Q 1.48 0.135 910 300 G =C + F6G/N7W/ 2.97 0.099 333 150 K8M/E9R H = C + N12R 3.08 0.094 310 180I = C + N12R/R13A 1.97 0.125 630 260 J = C + Q33K/K36R 2.00 0.073 370140 Exoface-1 plus Intraface-2 plus combined Interface-3 variants: K =D + F + H + J 5.04 0.030  60 180 L = E + F + H + J 4.87 0.030  62  60 M= F6-D38 of L 4.60 0.020  43 ND Kinetic measurements were determined ona BIAcore where a monoclonal IgG₁ was immobilized on the biosensor chip.The k_(on) values were determined by measuring k_(s) at 5 differentconcentrations, 4 μM, 3 μM, 2 μM, 1 μM, and 0.5 μM, and then plottingthe K_(s) values as a function of concentration. Standard error valueswere less than 2.5%. The k_(off) values were measured in duplicate withsaturating injections at a concentration of 25 μM. Reported values are#averaged, standard errors were less than 3.5%. The K_(d) values arecalculated from k_(off)/k_(on). Variant M is a synthetic peptideprepared by standard solid phase synthesis usingN-fluorenyl-methoxycarbonyl protocols.

The Intraface Selectants: Beginning with the consensus Exoface-1 peptideas a template, the Interface library was generated by mutating thehydrophobic core between helix-1 and helix-2 (Ala13, Ile17, Arg28,Ile32, and Leu35; FIG. 2B). Three of the five residues converged to anon-wild-type solution (A13R, I17A, and L35I) as shown in Table 1 above.These three residues form a cluster and apparently repack the core atthe open end of the two helices (FIG. 2B). Positions 13 and 17 are nextto each other on helix-1; the Ala/Ile wild-type combination is replacedby an Arg/Ala pair. Phage ELISA (Table 2 above) indicated that theconsensus sequence of A13R/I17A/L35I possessed an EC₅₀ that is about10-fold greater than that of consensus Exoface-1 variant.

The Interface Selectants: Beginning with the improved Exoface/Intrafaceselectants a template, five Interface libraries were generated bymutating nineteen residues, in groups of four, at or near the interfacebetween the Z-domain and the F_(c) portion of IgG (FIG. 2C). For theInterface-3A library, a weak consensus was found where Asn4 and Phe6were largely conserved as shown in Table 1 above.

The Interface-3B library generated two different consensus sequences asshown in Table 1 above; one conserved Phe6 and Asn7 while the othermutated these residues to Gly6 and Trp7. The aliphatic portion of Lys8in the wild-type sits at the helical intraface and does not make directcontact with the IgG; this position showed a strong consensus for Met.Concomitant with the K8M change, the negatively charged Glu9 wasneutralized to Gln or inverted to Lys (FIG. 2C).

From the Interface-3C library, it was determined that the contactresidues Gln10 and Gln11 were completely conserved. Asn12 was frequentlyconverted to a charged residue (Arg or Glu), while Arg13 was mostlyconserved. The four residues in the Interface-3D library (R14, Y15, L18,H19) were completely conserved, indicating that these residues cannot beimproved upon with natural amino acids. In the Interface-3E library, twonew consensus residues were determined in which Gln33 was replaced byLys, and Lys36 was replaced by Arg (FIG. 2F). Phage ELISA's for theconsensus selectants from each of these libraries (Table 2 above) showedimprovements in affinity ranging from two to three-fold over thestarting Exoface/Intraface variant.

Improvements in binding kinetics and affinities for the most preferredpeptides: The binding kinetics and affinities for each of the purifiedconsensus peptides were determined with respect to a monoclonal IgG₁ bysurface plasmon resonance as shown in Table 2 above. The starting38-residue peptide did not show any detectable binding at concentrationsup to 25 μM. The binding affinity of the combined Exoface/Intrafaceselectant (Table 2, variant C) represents an approximately 1000-foldimprovement over the starting 38 residue peptide. Variant C had a k_(on)that was equivalent to the full-length Z-domain, but a k_(off) that wasapproximately 100-fold greater.

The peptides derived from the Interface-3 libraries showed slightimprovements in k_(on) and/or k_(off), as shown in the data for variantsD through J in Table 2 above. Overall, each of these peptides showed atwo to three-fold improvement in affinity over variant C based on acomparison of relative K_(d) values. To further improve affinity, theconsensus variants from the Interface libraries (variants K and L, inTable 2 above) were combined. The resulting peptides showed twofold-improvements in k_(on) and five-fold improvements in k_(off)relative to most of the selectants in any of the Interface-3 libraries.The affinities for these mutants are only 6-fold weaker than thefull-length Z-domain and represent an improvement of greater than10⁴-fold over the starting 38-residue peptide. These final derivativesassociate about 2.5-times faster than the full-length Z-domain anddissociate only about 14-fold faster, indicating that the activity ofthe binding determinants on the analog is approaching that of the fulllength Z-domain. A synthetic peptide derived from variant L but with theN-terminal five residues deleted (Phe6-Asp38, variant M) actually has aslower k_(off) than variant L and thus a K_(d) value only four-foldhigher than the full-length Z-domain as shown in Table 2 above.Surprisingly, this peptide has only about 50% of the size (33 residues)of the original 59 residue Z-domain.

Evolving binding affinity in the two-helix derivative increases theα-helical structure: The secondary structural characteristics of some ofthese peptides after various stages of affinity optimization wereevaluated by CD spectroscopy (FIG. 2). The starting 38-residue peptideshowed only 11% helical content. However, the helical contentprogressively increased in going from this to the Exoface/Intraface mostpreferred variant (50%), and then to the final combined Interface mutant(56%). This compares with a maximum helical content of 63% estimatedfrom the number of residues in a helical conformation as determined fromthe x-ray coordinates (Deisenhofer, J., supra) of the two-helix segmentpresent in the intact B-domain.

Additional evidence suggests that the evolved two-helix bundle is highlystructured. First, the k_(on) values are comparable to or greater thanthe full-length Z-domain, indicating that little reorganization isnecessary. Many of the residues that are selected in either the Exoface,Intraface, or Interface libraries are buried in the two-helix bundlemodel (FIG. 2). It is likely that these residues were selected becausethey stabilize the core of the two-helix structure. Many of the residuesthat were absolutely conserved in the Interface libraries are highlyburied in the complex with the IgG, indicating that determinants fromthe two-helix motif are the same ones used in the full-length Z-domain.This was confirmed by alanine-scanning mutagenesis of the two-helixvariant L which shows that these conserved residues are critical forbinding. Lastly, binding from a discontinuous epitope usually depends onprecise display of determinants and therefore requires a highly orderedstructure. Preliminary results from the structural characterization of atwo helix variant by NMR confirm that this peptide adopts essentiallythe same conformation as helix-1 and helix-2 in the x-ray structure(Deisenhofer, J., supra).

Additionally, the alanine-scanning mutagenesis data for the variant Lpeptide indicated that an Arg12Ala substitution improved the IgG-bindingaffinity of the peptide.

EXAMPLE 2 Materials and Methods

A disulfide-linked 2 helix variant of the Z-domain was designed byincorporating a cysteine residue at the C-terminus and replacingglutamine 10 with cysteine in the peptide sequence of variant M inExample 1 above. A disulfide bond can be formed to covalently link the Nand C terminus. The design was modeled in the context of both the2-helix peptides of Example 1 above and the full length Z-domain.

The designed peptide, sequence FNMQCQRRFYEALHDPNLNEEQRNAKIKSIRDDC (SEQID NO:36), was synthesized using standard N-fluorenyl-methoxycarbonylprotocols on a solid support according to the method of Fields andNoble, Int. J. Peptide Protein Res., 35: 161-214 (1990). The peptide wassynthesized and cleaved with the cysteine thiols in the reduced form.Oxidation to form the disulfide linkage was performed by raising the pHof a cold (40° C.) aqueous solution of the peptide to 8.5 with ammoniumhydroxide, followed by the addition of a dilute aqueous solution ofpotassium ferricyanide until a persistant pale yellow color wasobtained. Alternatively, the cyclization of the peptide can be performedat a somewhat slower rate under the same conditions in the absence ofpotassium ferricyanide.

RESULTS AND DISCUSSION

The thermal stability of the disulfide linked peptide was assessed bycircular dichroism, monitoring Θ₂₂₂ as a measure of α-helicity. It wasdetermined that the disulfide linkage contributes significantly to thestability of the peptide structure, and that the peptide retained itshelical conformation even at 75° C. This conformational stability iscomparable to that of the native 3-helix Z-domain. Additional evidenceof the enhanced structure of the disulfide linked peptide was obtainedby NMR characterization.

The affinity of the disulfide linked peptide for IgG was determined bysurface plasmon resonance. The cyclized peptide was found to possess avery fast k_(on) rate of reaction for forming a complex with IgG(1.2×10⁶ sec⁻¹), approximately 6 fold faster than the native 3-helixZ-domain and about 2.5 fold faster than the nonlinked 2-helix peptide(variant M in Example 1 above). This increased k_(on) indicates that thedisulfide linked peptide is better organized for binding IgG. The rateof reaction for dissociation of the disulfide linked peptide/IgG complex(k_(off)) was determined to be about 30% slower than the nonlinked2-helix peptide (variant M) but still 7 fold faster than the nativeZ-domain. Overall, the enhanced binding kinetics of the disulfide linkedpeptide yield a dissocation constant (K_(d)) of 11 nM. This valuecompares quite favorably with the K_(d) of 10 nM for the nativeZ-domain. While the kinetics of IgG binding differ between the disulfidelinked peptide and the wild type Z domain, the net K_(d) is essentiallythe same.

47 33 amino acids Amino Acid Linear unknown 1 Xaa Xaa Xaa Xaa Gln GlnXaa Xaa Phe Tyr Glu Ala Leu His Asp 1 5 10 15 Pro Asn Leu Asn Glu GluGln Arg Asn Ala Lys Ile Xaa Ser Ile 20 25 30 Xaa Asp Asp 33 5 aminoacids Amino Acid Linear unknown 2 Ala Val Xaa Xaa Xaa 1 5 34 amino acidsAmino Acid Linear unknown 3 Xaa Xaa Xaa Xaa Cys Gln Xaa Xaa Phe Tyr GluAla Leu His Asp 1 5 10 15 Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala LysIle Xaa Ser Ile 20 25 30 Xaa Asp Asp Cys 34 59 amino acids Amino AcidLinear unknown 4 Ala Val Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala PheTyr 1 5 10 15 Glu Ile Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg AsnAla 20 25 30 Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu35 40 45 Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys 50 5559 38 amino acids Amino Acid Linear unknown 5 Ala Val Asp Asn Lys PheAsn Lys Glu Gln Gln Asn Arg Phe Tyr 1 5 10 15 Glu Ala Leu His Asp ProAsn Leu Asn Glu Glu Gln Arg Asn Ala 20 25 30 Lys Ile Gln Ser Ile Lys AspAsp 35 38 38 amino acids Amino Acid Linear unknown 6 Ala Val Arg Asn GlyPhe Asn Lys Glu Gln Gln Asn Arg Phe Tyr 1 5 10 15 Glu Ala Leu His AspPro Asn Leu Asn Glu Glu Gln Arg Asn Ala 20 25 30 Lys Ile Gln Ser Ile LysAsp Asp 35 38 38 amino acids Amino Acid Linear unknown 7 Ala Val Ala GlnSer Phe Asn Lys Glu Gln Gln Asn Arg Phe Tyr 1 5 10 15 Glu Ala Leu HisAsp Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala 20 25 30 Lys Ile Gln Ser IleLys Asp Asp 35 38 38 amino acids Amino Acid Linear unknown 8 Ala Val AspAsn Lys Phe Asn Met Gln Gln Gln Asn Arg Phe Tyr 1 5 10 15 Glu Ala LeuHis Asp Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala 20 25 30 Lys Ile Gln SerIle Lys Asp Asp 35 38 38 amino acids Amino Acid Linear unknown 9 Ala ValAsp Asn Lys Gly Trp Met Arg Gln Gln Asn Arg Phe Tyr 1 5 10 15 Glu AlaLeu His Asp Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala 20 25 30 Lys Ile GlnSer Ile Lys Asp Asp 35 38 38 amino acids Amino Acid Linear unknown 10Ala Val Asp Asn Lys Phe Asn Lys Glu Gln Gln Arg Arg Phe Tyr 1 5 10 15Glu Ala Leu His Asp Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala 20 25 30 LysIle Gln Ser Ile Lys Asp Asp 35 38 38 amino acids Amino Acid Linearunknown 11 Ala Val Asp Asn Lys Phe Asn Lys Glu Gln Gln Arg Ala Phe Tyr 15 10 15 Glu Ala Leu His Asp Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala 2025 30 Lys Ile Gln Ser Ile Lys Asp Asp 35 38 38 amino acids Amino AcidLinear unknown 12 Ala Val Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn ArgPhe Tyr 1 5 10 15 Glu Ala Leu His Asp Pro Asn Leu Asn Glu Glu Gln ArgAsn Ala 20 25 30 Lys Ile Lys Ser Ile Arg Asp Asp 35 38 38 amino acidsAmino Acid Linear unknown 13 Ala Val Arg Asn Gly Phe Asn Met Gln Gln GlnArg Arg Phe Tyr 1 5 10 15 Glu Ala Leu His Asp Pro Asn Leu Asn Glu GluGln Arg Asn Ala 20 25 30 Lys Ile Lys Ser Ile Arg Asp Asp 35 38 38 aminoacids Amino Acid Linear unknown 14 Ala Val Ala Gln Ser Phe Asn Met GlnGln Gln Arg Arg Phe Tyr 1 5 10 15 Glu Ala Leu His Asp Pro Asn Leu AsnGlu Glu Gln Arg Asn Ala 20 25 30 Lys Ile Lys Ser Ile Arg Asp Asp 35 3833 amino acids Amino Acid Linear unknown 15 Phe Asn Met Gln Gln Gln ArgArg Phe Tyr Glu Ala Leu His Asp 1 5 10 15 Pro Asn Leu Asn Glu Glu GlnArg Asn Ala Lys Ile Lys Ser Ile 20 25 30 Arg Asp Asp 33 38 amino acidsAmino Acid Linear unknown 16 Ala Val Asp Asn Lys Phe Asn Lys Glu Gln GlnAla Arg Phe Tyr 1 5 10 15 Glu Ala Leu His Asp Pro Asn Leu Asn Glu GluGln Arg Asn Ala 20 25 30 Lys Ile Gln Ser Ile Lys Asp Asp 35 38 38 aminoacids Amino Acid Linear unknown 17 Ala Val Arg Asn Gly Phe Asn Lys GluGln Gln Ala Arg Phe Tyr 1 5 10 15 Glu Ala Leu His Asp Pro Asn Leu AsnGlu Glu Gln Arg Asn Ala 20 25 30 Lys Ile Gln Ser Ile Lys Asp Asp 35 3838 amino acids Amino Acid Linear unknown 18 Ala Val Ala Gln Ser Phe AsnLys Glu Gln Gln Ala Arg Phe Tyr 1 5 10 15 Glu Ala Leu His Asp Pro AsnLeu Asn Glu Glu Gln Arg Asn Ala 20 25 30 Lys Ile Gln Ser Ile Lys Asp Asp35 38 38 amino acids Amino Acid Linear unknown 19 Ala Val Asp Asn LysPhe Asn Met Gln Gln Gln Ala Arg Phe Tyr 1 5 10 15 Glu Ala Leu His AspPro Asn Leu Asn Glu Glu Gln Arg Asn Ala 20 25 30 Lys Ile Gln Ser Ile LysAsp Asp 35 38 38 amino acids Amino Acid Linear unknown 20 Ala Val AspAsn Lys Gly Trp Met Arg Gln Gln Ala Arg Phe Tyr 1 5 10 15 Glu Ala LeuHis Asp Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala 20 25 30 Lys Ile Gln SerIle Lys Asp Asp 35 38 38 amino acids Amino Acid Linear unknown 21 AlaVal Asp Asn Lys Phe Asn Lys Glu Gln Gln Ala Ala Phe Tyr 1 5 10 15 GluAla Leu His Asp Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala 20 25 30 Lys IleGln Ser Ile Lys Asp Asp 35 38 38 amino acids Amino Acid Linear unknown22 Ala Val Asp Asn Lys Phe Asn Lys Glu Gln Gln Ala Arg Phe Tyr 1 5 10 15Glu Ala Leu His Asp Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala 20 25 30 LysIle Lys Ser Ile Arg Asp Asp 35 38 38 amino acids Amino Acid Linearunknown 23 Ala Val Arg Asn Gly Phe Asn Met Gln Gln Gln Ala Arg Phe Tyr 15 10 15 Glu Ala Leu His Asp Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala 2025 30 Lys Ile Lys Ser Ile Arg Asp Asp 35 38 38 amino acids Amino AcidLinear unknown 24 Ala Val Ala Gln Ser Phe Asn Met Gln Gln Gln Ala ArgPhe Tyr 1 5 10 15 Glu Ala Leu His Asp Pro Asn Leu Asn Glu Glu Gln ArgAsn Ala 20 25 30 Lys Ile Lys Ser Ile Arg Asp Asp 35 38 33 amino acidsAmino Acid Linear unknown 25 Phe Asn Met Gln Gln Gln Ala Arg Phe Tyr GluAla Leu His Asp 1 5 10 15 Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala LysIle Lys Ser Ile 20 25 30 Arg Asp Asp 33 39 amino acids Amino Acid Linearunknown 26 Ala Val Asp Asn Lys Phe Asn Lys Glu Cys Gln Asn Arg Phe Tyr 15 10 15 Glu Ala Leu His Asp Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala 2025 30 Lys Ile Gln Ser Ile Lys Asp Asp Cys 35 39 39 amino acids AminoAcid Linear unknown 27 Ala Val Arg Asn Gly Phe Asn Lys Glu Cys Gln AsnArg Phe Tyr 1 5 10 15 Glu Ala Leu His Asp Pro Asn Leu Asn Glu Glu GlnArg Asn Ala 20 25 30 Lys Ile Gln Ser Ile Lys Asp Asp Cys 35 39 39 aminoacids Amino Acid Linear unknown 28 Ala Val Ala Gln Ser Phe Asn Lys GluCys Gln Asn Arg Phe Tyr 1 5 10 15 Glu Ala Leu His Asp Pro Asn Leu AsnGlu Glu Gln Arg Asn Ala 20 25 30 Lys Ile Gln Ser Ile Lys Asp Asp Cys 3539 39 amino acids Amino Acid Linear unknown 29 Ala Val Asp Asn Lys PheAsn Met Gln Cys Gln Asn Arg Phe Tyr 1 5 10 15 Glu Ala Leu His Asp ProAsn Leu Asn Glu Glu Gln Arg Asn Ala 20 25 30 Lys Ile Gln Ser Ile Lys AspAsp Cys 35 39 39 amino acids Amino Acid Linear unknown 30 Ala Val AspAsn Lys Gly Trp Met Arg Cys Gln Asn Arg Phe Tyr 1 5 10 15 Glu Ala LeuHis Asp Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala 20 25 30 Lys Ile Gln SerIle Lys Asp Asp Cys 35 39 39 amino acids Amino Acid Linear unknown 31Ala Val Asp Asn Lys Phe Asn Lys Glu Cys Gln Arg Arg Phe Tyr 1 5 10 15Glu Ala Leu His Asp Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala 20 25 30 LysIle Gln Ser Ile Lys Asp Asp Cys 35 39 39 amino acids Amino Acid Linearunknown 32 Ala Val Asp Asn Lys Phe Asn Lys Glu Cys Gln Arg Ala Phe Tyr 15 10 15 Glu Ala Leu His Asp Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala 2025 30 Lys Ile Gln Ser Ile Lys Asp Asp Cys 35 39 39 amino acids AminoAcid Linear unknown 33 Ala Val Asp Asn Lys Phe Asn Lys Glu Cys Gln AsnArg Phe Tyr 1 5 10 15 Glu Ala Leu His Asp Pro Asn Leu Asn Glu Glu GlnArg Asn Ala 20 25 30 Lys Ile Lys Ser Ile Arg Asp Asp Cys 35 39 39 aminoacids Amino Acid Linear unknown 34 Ala Val Arg Asn Gly Phe Asn Met GlnCys Gln Arg Arg Phe Tyr 1 5 10 15 Glu Ala Leu His Asp Pro Asn Leu AsnGlu Glu Gln Arg Asn Ala 20 25 30 Lys Ile Lys Ser Ile Arg Asp Asp Cys 3539 39 amino acids Amino Acid Linear unknown 35 Ala Val Ala Gln Ser PheAsn Met Gln Cys Gln Arg Arg Phe Tyr 1 5 10 15 Glu Ala Leu His Asp ProAsn Leu Asn Glu Glu Gln Arg Asn Ala 20 25 30 Lys Ile Lys Ser Ile Arg AspAsp Cys 35 39 34 amino acids Amino Acid Linear unknown 36 Phe Asn MetGln Cys Gln Arg Arg Phe Tyr Glu Ala Leu His Asp 1 5 10 15 Pro Asn LeuAsn Glu Glu Gln Arg Asn Ala Lys Ile Lys Ser Ile 20 25 30 Arg Asp Asp Cys34 39 amino acids Amino Acid Linear unknown 37 Ala Val Asp Asn Lys PheAsn Lys Glu Cys Gln Ala Arg Phe Tyr 1 5 10 15 Glu Ala Leu His Asp ProAsn Leu Asn Glu Glu Gln Arg Asn Ala 20 25 30 Lys Ile Gln Ser Ile Lys AspAsp Cys 35 39 39 amino acids Amino Acid Linear unknown 38 Ala Val ArgAsn Gly Phe Asn Lys Glu Cys Gln Ala Arg Phe Tyr 1 5 10 15 Glu Ala LeuHis Asp Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala 20 25 30 Lys Ile Gln SerIle Lys Asp Asp Cys 35 39 39 amino acids Amino Acid Linear unknown 39Ala Val Ala Gln Ser Phe Asn Lys Glu Cys Gln Ala Arg Phe Tyr 1 5 10 15Glu Ala Leu His Asp Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala 20 25 30 LysIle Gln Ser Ile Lys Asp Asp Cys 35 39 39 amino acids Amino Acid Linearunknown 40 Ala Val Asp Asn Lys Phe Asn Met Gln Cys Gln Ala Arg Phe Tyr 15 10 15 Glu Ala Leu His Asp Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala 2025 30 Lys Ile Gln Ser Ile Lys Asp Asp Cys 35 39 39 amino acids AminoAcid Linear unknown 41 Ala Val Asp Asn Lys Gly Trp Met Arg Cys Gln AlaArg Phe Tyr 1 5 10 15 Glu Ala Leu His Asp Pro Asn Leu Asn Glu Glu GlnArg Asn Ala 20 25 30 Lys Ile Gln Ser Ile Lys Asp Asp Cys 35 39 39 aminoacids Amino Acid Linear unknown 42 Ala Val Asp Asn Lys Phe Asn Lys GluCys Gln Ala Ala Phe Tyr 1 5 10 15 Glu Ala Leu His Asp Pro Asn Leu AsnGlu Glu Gln Arg Asn Ala 20 25 30 Lys Ile Gln Ser Ile Lys Asp Asp Cys 3539 39 amino acids Amino Acid Linear unknown 43 Ala Val Asp Asn Lys PheAsn Lys Glu Cys Gln Ala Arg Phe Tyr 1 5 10 15 Glu Ala Leu His Asp ProAsn Leu Asn Glu Glu Gln Arg Asn Ala 20 25 30 Lys Ile Lys Ser Ile Arg AspAsp Cys 35 39 39 amino acids Amino Acid Linear unknown 44 Ala Val ArgAsn Gly Phe Asn Met Gln Cys Gln Ala Arg Phe Tyr 1 5 10 15 Glu Ala LeuHis Asp Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala 20 25 30 Lys Ile Lys SerIle Arg Asp Asp Cys 35 39 39 amino acids Amino Acid Linear unknown 45Ala Val Ala Gln Ser Phe Asn Met Gln Cys Gln Ala Arg Phe Tyr 1 5 10 15Glu Ala Leu His Asp Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala 20 25 30 LysIle Lys Ser Ile Arg Asp Asp Cys 35 39 34 amino acids Amino Acid Linearunknown 46 Phe Asn Met Gln Cys Gln Ala Arg Phe Tyr Glu Ala Leu His Asp 15 10 15 Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Lys Ile Lys Ser Ile 2025 30 Arg Asp Asp Cys 34 38 amino acids Amino Acid Linear unknown 47 AlaVal Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr 1 5 10 15 GluIle Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala 20 25 30 Phe IleGln Ser Leu Lys Asp Asp 35 38

We claim:
 1. A compound represented by Formula (II):

where X₁ is selected from the group consisting of H, C₁-C₆alkanoyl, and Z-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2); where Z is selected from the group consisting of H and C₁-C₆alkanoyl; AA₃ is selected from the group consisting of Asp, Arg, and Ala; AA₄ is selected from the group consisting of Asn and Gln; and AA₅is selected from the group consisting of Lys, Gly, and Ser; AA₆ is selected from the group consisting of Phe and Gly; AA₇ is selected from the group consisting of Asn and Trp; AA₈ is selected from the group consisting of Lys and Met; AA₉ is selected from the group consisting of Glu, Gln, and Arg; AA₁₂ is selected from the group consisting of Asn, Ala, and Arg; AA₁₃ is selected from the group consisting of Ala and Arg; AA₃₃ is selected from th e group consisting of Gln and Lys; AA₃₆ is selected from the group consisting of Lys and Arg; and X₂ is selected from the group consisting of OR₁ and NR₁R₂ where R₁ and R₂ are independently selected from the group consisting of H, C₁-C₆alkyl, C₆-C₁₂aryl and C₆-C₁₂aryl-C₁-C₆alkyl; which compound is covalently linked to a macromolecule to form a conjugate.
 2. The conjugate of claim 1 wherein the macromolecule is a solid support.
 3. The conjugate of claim 1 wherein the compound is selected from the group consisting of:

where X₁ is selected from the group consisting of H and C₁-C₆alkanoyl; Z is selected from the group consisting of H and C₁-C₆alkanoyl; and X₂ is selected from the group consisting of OR₁ and NR₁R₂ where R₁ and R₂ are independently selected from the group consisting of H, C₁-C₆alkyl, C₆-C₁₂aryl and C₆-C₁₂aryl-C₁-C₆alkyl.
 4. The conjugate of claim 1 wherein the compound is selected from the group consisting of:

where X₁ is selected from the group consisting of H and C₁-C₆alkanoyl; Z is selected from the group consisting of H and C₁-C₆alkanoyl; and X₂ is selected from the group consisting of OR₁ and NR₁R₂ where R₁ and R₂ are independently selected from the group consisting of H, C₁-C₆alkyl, C₆-C₁₂aryl and C₆-C₁₂aryl-C₁-C₆alkyl.
 5. A compound represented by Formula (II):

where X₁ is selected from the group consisting of H, C₁-C₆alkanoyl, and Z-Ala-Val-AA₃-AA₄-AA₅ (SEQ ID NO:2); where Z is selected from the group consisting of H and C₁-C₆alkanoyl; AA₃ is selected from the group consisting of Asp, Arg, and Ala; AA₄ is selected from the group consisting of Asn and Gln; and AA₅ is selected from the group consisting of Lys, Gly, and Ser; AA₆ is selected from the group consisting of Phe and Gly; AA₇ is selected from the group consisting of Asn and Trp; AA₈ is selected from the group consisting of Lys and Met; AA₉ is selected from the group consisting of Glu, Gln, and Arg; AA₁₂ is selected from the group consisting of Asn, Ala, and Arg; AA₁₃ is selected from the group consisting of Ala and Arg; AA₃₃ is selected from the group consisting of Gln and Lys; AA₃₆ is selected from the group consisting of Lys and Arg; and X₂ is selected from the group consisting of OR₁ and NR₁R₂ where R₁ and R₂ are independently selected from the group consisting of H, C₁-C₆alkyl, C₆-C₁₂aryl and C₆-C₁₂aryl-C₁-C₆alkyl; which compound is fused to a selected polypeptide to form a fusion protein.
 6. The fusion protein of claim 5 wherein the compound of Formula (II) is specifically cleavable from the amino acid sequence of the selected polypeptide.
 7. The fusion protein of claim 5 wherein the compound is selected from the group consisting of:

where X₁ is selected from the group consisting of H and C₁-C₆alkanoyl; Z is selected from the group consisting of H and C₁-C₆alkanoyl; and X₂ is selected from the group consisting of OR₁ and NR₁R₂ where R₁ and R₂ are independently selected from the group consisting of H, C₁-C₆alkyl, C₆-C₁₂aryl and C₆-C₁₂aryl-C₁-C₆alkyl.
 8. The fusion protein of claim 5 wherein the compound is selected from the group consisting of:

where X₁ is selected from the group consisting of H and C₁-C₆alkanoyl; Z is selected from the group consisting of H and C₁-C₆alkanoyl; and X₂ is selected from the group consisting of OR₁ and NR₁R₂ where R₁ and R₂ are independently selected from the group consisting of H, C₁-C₆alkyl, C₆-C₁₂aryl and C₆-C₁₂aryl-C₁-C₆alkyl. 